Single nucleotide polymorphisms (snp) and association with resistance to immune tolerance induction

ABSTRACT

This application discloses methods, systems and kits for correlating the presence or absence of certain nucleic acid sequences within a population with the ability to create immune tolerance in that same population. Tolerance can be induced by solo or repeated administration of antigen, including soluble antigens administered either intravenously or sublingually. This application also discloses methods for detecting variants. In addition the application addresses the use or avoidance of non steroidal anti inflammatory drugs in therapy.

FIELD OF THE INVENTION

The present invention is in the field of the immune system and therapy.In particular, the present invention relates to specific nucleic acidsequences in the human genome, and their association with diseases andpathologies as well as immune tolerance induction. Based on differencesin allele frequencies in the patient population relative to normalindividuals, the naturally-occurring nucleic acid sequences disclosedherein can be used as targets for the design of diagnostic reagents andthe development of therapeutic agents, as well as for diseaseassociation and linkage analysis. In particular, the nucleic acidsequences of the present invention are useful for identifying anindividual (e.g., patient) who is at an increased or decreased risk ofdeveloping a disease or pathology and for early detection of the diseaseor pathology, for providing clinically important information for theprevention and/or treatment of disease or pathology, and for screeningand selecting therapeutic agents. Further, the presence or absence ofone or more nucleic acid sequences may be used to determine the type anddose of antigen given to induce immune tolerance. One or more antigensmay be administered to a patient having or not having a particularnucleic acid sequence, where the antigen may induce immune tolerance ina patient. The nucleic acid sequences disclosed herein are also usefulfor human identification applications. Methods, assays, kits, andreagents for detecting the presence of these polymorphisms and theirencoded products are also provided. In addition, the invention addressesthe use or absence of non-steroidal anti-inflammatory drugs in therapy.

BACKGROUND

The genomes of all organisms undergo spontaneous mutation in the courseof their continuing evolution, generating variant forms of progenitorgenetic sequences. A variant form may confer an evolutionary advantageor disadvantage relative to a progenitor form or may be neutral. In someinstances, a variant form confers an evolutionary advantage to thespecies and is eventually incorporated into the DNA of many or mostmembers of the species and effectively becomes the progenitor form.Additionally, the effects of a variant form may be both beneficial anddetrimental, depending on the circumstances. For example, a heterozygoussickle cell mutation confers resistance to malaria, but a homozygoussickle cell mutation is usually lethal. In many cases, both progenitorand variant forms survive and co-exist in a species population. Thecoexistence of multiple forms of a genetic sequence gives rise togenetic polymorphisms, including single nucleotide polymorphisms,otherwise known as “SNPs”. SNPs can also arise in areas of the genomewith no apparent function, but the SNP can be genetically linked to avariant sequence in the genome. Thus, the SNP can closely correlate withthe variant sequence of the genome, depending on how close the geneticlinkage is.

Approximately 90% of all polymorphisms in the human genome are SNPs.SNPs are single base positions in DNA at which different alleles, oralternative nucleotides, exist in a population. The SNP position(interchangeably referred to herein as SNP, SNP site, or SNP locus) isusually preceded by and followed by highly conserved sequences of theallele (e.g., sequences that vary in less than 1/100 or 1/1000 membersof the populations). An individual may be homozygous or heterozygous foran allele at each SNP position.

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. Some believe the patientresponse is due to their genetic make-up, which results in havingaltered receptors, enzymes, or some change in cell physiology. Thus, thedifference in genetic make-up results in a different response fromothers in the population. As such, researchers have expressed hope thatthe nucleic acid sequences of a patient can be used in pharmaceuticalresearch to assist the drug development and selection process. To date,it is our belief that use of the nucleic acid sequence of a patient inpharmaceuticals has not been applied to immune tolerance.

Immune or immunological tolerance is the process by which the immunesystem does not mount an immune response to an otherwise immunogenicantigen, or has its immune response redirected in a suppressive manner.Acquired or induced tolerance refers to the immune system's adaptationto external antigens characterized by a specific non-reactivity of thelymphoid tissues to a given antigen that in other circumstances wouldlikely induce cell-mediated or humoral immunity. One of the mostimportant natural kinds of acquired tolerance occurs during pregnancy,where the fetus and the placenta must be tolerated by the maternalimmune system. There are numerous models for the induction of tolerance,including use of the eutherian fetoembryonic defense system andinduction of tolerance primarily requires the participation ofregulatory T cells.

One specific type of immune tolerance, oral tolerance, is the specificsuppression of cellular and/or humoral immune reactivity to an antigenby prior administration of the antigen by the oral route, probablyevolved to prevent hypersensitivity reactions to food proteins andbacterial antigens present in the mucosal flora. It is of immenseimmunological importance, since it is a continuous natural immunologicevent driven by exogenous antigen. Due to their privileged access to theinternal milieu, antigens that continuously contact the mucosa representa frontier between foreign and self components. Oral tolerance evolvedto treat external agents that gain access to the body via a naturalroute as internal components without danger signals, which then becomepart of self. Failure of oral tolerance is attributed to the developmentand pathogenesis of several immunologically based diseases, includingInflammatory Bowel Disease (Crohn's Disease and Ulcerative Colitis).Other common forms of tolerance induction contemplated in this inventioninclude internasel and IV tolerance induction.

Today genomics is still not used to refine our medical management,despite the large quantities of research into the genome and SNPs. Assuch, there are few reliable wide-scale assays, kits and methods thatexamine an individual's genome to find inherited susceptibility, geneexpression, and predicted pharmacogenomic response. More specifically,there is no use of genomics in the field of immune tolerance.

The inventors are credited with providing methods, reagents, kits andassays that merge genomics with immune tolerance. As such, the inventorsare able to detect nucleic acid sequences, and based on the presenceand/or absence of nucleic acid sequences; tailor a method of care forthe patient who desires induction of immune tolerance. In one specificembodiment, the nucleic acid sequences of interest are SNPs.

SUMMARY OF THE INVENTION

The present invention relates to correlating the presence or absence ofcertain nucleic acid sequences within a population with the ability tocreate immune tolerance in that same population. This correlation may beused to assist with induction of immune tolerance. In some embodiments,the invention concerns tolerance induced by repeated administration ofvery large doses of antigen, or of small doses that are below thethreshold required for stimulation of an immune response. In someembodiments, tolerance is most readily induced by soluble antigensadministered either intravenously or sublingually. Furthermore, it'scontemplated that immunosuppression also facilitates the induction oftolerance. Based on the correlation of the presence or absence ofcertain nucleic acid sequences associated with specific diseases ordisorders, the present invention also provides for methods of detectingvariants. In one specific embodiment, the nucleic acid sequences ofinterest are SNPs. In addition embodiments of the invention address theuse or avoidance of non streroidal anti inflammatory drugs in therapy.

The invention includes a method for screening for susceptibility toimmune tolerance development, comprising screening for at least one SNP.The method of screening may include FISH, use of a DNA array, and/orhybridizing a polynucleotide probe.

The invention includes a method for screening for susceptibility toimmune tolerance development, including use of allele-specific probehybridization, allele-specific primer extension, allele-specificamplification, sequencing, 5′ nuclease digestion, molecular beaconassay, oligonucleotide ligation assay, size analysis, and/orsingle-stranded conformation polymorphism.

The invention includes a method for screening for susceptibility toimmune tolerance development, including correlating the presence orabsence of the at least one SNP with ability of a host to develop immunetolerance as a result of administration of one or more antigens ortherapeutic agents to the host. The antigen may be collagen, includingcollagen selected from the group types consisting of I, II, III, IV, V,VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX,XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII and XXVIII.

The invention includes a method for screening for susceptibility toimmune tolerance development, including obtaining a sample from saidhost comprising nucleic acid; isolating nucleic acid from said sample;assaying said sample for the presence or absence of at least one SNP,wherein the presence or absence of the at least one SNP is indicative ofan increased susceptibility to develop immune tolerance. The sample maybe whole blood, blood plasma, urine, tears, semen, saliva, buccalmucosa, interstitial fluid, lymph fluid, meningeal fluid, amnioticfluid, glandular fluid, sputum, feces, perspiration, mucous, vaginalsecretion, cerebrospinal fluid, hair, skin, fecal material, woundexudate, wound homogenate, and wound fluid. Further, the method mayinclude induction of immune tolerance by administration of at least oneantigen to the host, where the antigen can be a type of collagen.

The immune tolerance may be to any autoimmune disease such as scleroticdisease like systemic sclerosis.

The invention includes a method for screening for susceptibility toimmune tolerance development, including use of one or more computerprograms for use with at least one computer system, where computerprogram includes a plurality of instructions including at least oneinstruction for aiding in identification of the presence or absence ofsaid at least one SNP; at least one instruction for associating thepresence or absence of said at least one SNP with at least one diseasestate; and at least one instruction for correlating the presence orabsence of said at least one SNP with a score indicating susceptibilityof a host to develop immune tolerance. The computer may also generate areport including the results of the plurality of instruction, where thereport may be transmitted over a network, on-line portal, by paper ore-mail in a secure or non-secure manner.

The invention includes a method of administering at least onetherapeutic agent, the method comprising genotyping one or more SNP(s)in the nucleic acid of a host, correlating the one or more SNP(s) withone or more diseases or disorders, using a mathematical algorithm todetermine probability that said host will respond positively ornegatively to administration of at least one therapeutic agent, andadministrating or not administrating a therapeutic agent to the hostbased on the results of said mathematical algorithm.

The invention includes a method for conducting a clinical trial in whichone or more antigen(s) are evaluated, including genotyping one or moreSNP(s) relating to one or more diseases or disorders; analyzing thegenotyping results; determining a course of action based on the resultsof said genotyping, wherein said course of action comprises includingindividual in the clinical trial based on the results of said genotypinghaving indicated that said individuals are likely to respond to said oneor more antigen(s), and/or excluding individuals from participating inthe clinical trial based on the results of said genotyping havingindicated that said individuals are not likely to respond to said one ormore antigen(s).

The invention includes a method for identifying an individual who has analtered risk for developing an autoimmune disease, comprising detectinga single nucleotide polymorphism (SNP) in SEQ ID NO: 1 in saidindividual's nucleic acids, wherein the presence of the SNP iscorrelated with an altered risk for autoimmune disease.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a flow chart in accordance with some embodiments ofthe invention, demonstrating a process of determining if one or morenucleic acids is correlated with induction of immune tolerance.

FIG. 2 illustrates a flow chart in accordance with some embodiments ofthe invention, demonstrating a process of determining an individual'streatment regiment by the presence of absence of one or more nucleicacids.

FIG. 3 illustrates a flow chart in accordance with some embodiments ofthe invention, demonstrating a process of determining an individual'streatment regiment by the presence of absence of one or more nucleicacids.

FIG. 4 illustrates a flow chart in accordance with some embodiments ofthe invention, demonstrating a process of determining an individual'streatment regiment by the presence of absence of one or more nucleicacids.

FIG. 5 illustrates a flow chart in accordance with some embodiments ofthe invention, demonstrating a process of determining an individual'streatment regiment by the presence of absence of one or morepolypeptides.

FIG. 6 is a chart showing the percent of arthritic joints correlatedwith quantity of collagen II given by gavage to mice.

FIG. 7 is a chart showing abrogation of certain forms of oral toleranceby piroxicam.

FIG. 8 is a chart showing IFN-γ production by α1 (II) stimulated spleencells.

FIG. 9 is a chart showing COX-2 inhibitor SC'236 abrogates oraltolerance induction.

FIG. 10 is a chart showing persistent abrogation of oral tolerance bypiroxicam.

FIG. 11 is a chart showing the effect of piroxicam or CII on Peyer'spatch spleen co-culture.

FIG. 12 is a chart showing the effect of piroxicam or CII on mesentericlymph node cell proliferation

FIG. 13 is a chart showing the effect of oral collagen II treatment ofrheumatoid arthritis patients and how it modulates PBMC IFN productionby PBMC.

FIG. 14 is a chart the genotyping of persons with rheumatoid arthritisand their ability to induce immune tolerance.

FIG. 15 is a chart showing the markers and genes contain potential SNPsof interest.

FIG. 16 is a chart showing a significant reduction in MRSS at 12 monthsversus placebo patients.

DEFINITIONS

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions. To assist in describing theinvention, the following definitions are provided.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides in either single- or double-stranded form. Those skilledin the art will readily recognize that reference to a particular site onone strand refers, as well, to the corresponding site on a complementarystrand. Unless specifically limited, the term encompasses nucleic acidscontaining known analogues of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues. The termnucleic acid is used interchangeably with “nucleic acid sequence,”“gene,” “cDNA,” and “mRNA” encoded by a gene.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, α-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either the commonly known three letter symbols orby the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes.

The term “genotype” as used herein broadly refers to the geneticcomposition of an organism, including, for example, whether a diploidorganism is heterozygous or homozygous for one or more variant allelesof interest.

As used herein, references to “SNPs” and “SNP genotypes” includeindividual SNPs and/or haplotypes, which are groups of SNPs that aregenerally inherited together. Haplotypes can have stronger correlationswith diseases or other phenotypic effects compared with individual SNPs,and therefore may provide increased diagnostic accuracy in some cases.

The SNPs of the current invention may arise from a substitution of oneor more nucleotides for another at the polymorphic site. Substitutionscan be transitions or transversions. A transition is the replacement ofone purine nucleotide by another purine nucleotide, or one pyrimidine byanother pyrimidine. A transversion is the replacement of a purine by apyrimidine, or vice versa. A SNP may also be a single base insertion ordeletion variant.

The SNPs of the current invention may arise from a synonymous codonchange, or silent mutation/SNP (terms such as “SNP”, “polymorphism”,“mutation”, “mutant”, “variation”, and “variant” are used hereininterchangeably), is one that does not result in a change of amino aciddue to the degeneracy of the genetic code. A substitution that changes acodon coding for one amino acid to a codon coding for a different aminoacid (i.e., a non-synonymous codon change) is referred to as a missensemutation. A nonsense mutation results in a type of non-synonymous codonchange in which a stop codon is formed, thereby leading to prematuretermination of a polypeptide chain and a truncated protein. Aread-through mutation is another type of non-synonymous codon changethat causes the destruction of a stop codon, thereby resulting in anextended polypeptide product. SNPs may include all allelics, includingbi-, tri-, or tetra-allelics.

In defining a SNP position, SNP allele, or nucleotide sequence,reference to an adenine, a thymine (uridine), a cytosine, or a guanineat a particular site on one strand of a nucleic acid molecule alsodefines the thymine (uridine), adenine, guanine, or cytosine(respectively) at the corresponding site on a complementary strand ofthe nucleic acid molecule. Thus, reference may be made to either strandto refer to a particular SNP position, SNP allele, or nucleotidesequence. Probes and primers, may be designed to hybridize to eitherstrand and SNP genotyping methods disclosed herein may generally targeteither strand.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. References to “polypeptides,”“peptides” or “proteins” of the present invention include peptides,polypeptides, proteins, or fragments thereof, that contain at least oneamino acid residue that differs from the corresponding amino acidsequence of the art-known peptide/polypeptide/protein (the protein maybe interchangeably referred to as the “wild-type”, “reference”, or“normal” protein). Such variant peptides/polypeptides/proteins canresult from a codon change caused by a nonsynonymous nucleotidesubstitution at a protein-coding position (i.e., a missense mutation)disclosed by the present invention. Variantpeptides/polypeptides/proteins of the present invention can also resultfrom a nonsense mutation, i.e., a SNP that creates a premature stopcodon, a SNP that generates a read-through mutation by abolishing a stopcodon, or due to any SNP disclosed by the present invention thatotherwise alters the structure, function/activity, or expression of aprotein, such as a SNP in a regulatory region (e.g., a promoter orenhancer) or a SNP that leads to alternative or defective splicing, suchas a SNP in an intron or a SNP at an exon/intron boundary. As usedherein, the terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably.

For all embodiments, the terms “individual” and “host” are usedinterchangably, and are not limited to humans. According to an aspect ofthe present invention, the “individual” may be any vertebrate, includingmammals such as primates and including humans, dogs, cats, cows, goats,pigs, sheep, and monkeys. Thus, the disclosed invention may beapplicable to treatment of animals through, for example, animal water,animal feed, animal pharmaceuticals, and the like. According to anaspect of the present invention, the individual may be healthy orsuffering from a disease. In general, however, methods of the presentinvention can be effectively used if applied to a human who suffers froman auto-immunity disease or a disease caused by a pathogenicmicroorganism and a type of a certain nucleic acid sequence in theindividual.

The term “altered” may be used herein to encompass either an increasedor a decreased risk/likelihood.

The term “specific disease,” “disease” or “disorder” used hereinencompasses auto antibody diseases as well as diseases associated with apathogenic microorganism, such as for example, a virus, bacterium, yeastor mycoplasma as well as oncological diseases. However, the disease isnot particularly limited.

The term “practitioner” or “medical practitioner” used herein includesany person who engages in medicine or related medical arts as aprofession, including the medical, biotechnology or pharmaceuticalindustry.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of determining if an individualis likely to develop immune tolerance by detecting the alteredexpression (either higher or lower expression) or unique expression ofnucleic acid sequences, as well as the gene sequence in the individual,even if they are not expressed or only transiently expressed. As such,some embodiments provide methods to determine if an individual that hasthe ability to develop immune tolerance has a differential and uniqueexpression of known and unknown nucleic acid sequence from thoseindividuals who do not have the ability to develop immune tolerance. Insome embodiments, the nucleic acid sequences are SNPs.

The present invention also provides nucleic acid sequences, methods andreagents for detecting the nucleic acid sequences, uses of nucleic acidsequences in kits or assays for use in advance of inducing immunetolerance.

The invention contemplates use of nucleic acid sequences that areassociated with either an increased risk of having or developing immunetolerance, or a decreased risk of having or developing immune tolerance.The presence of certain nucleic acid sequences (or their mRNA or proteinencoded products) can be assayed to determine whether an individualpossesses a nucleic acid sequences that is indicative of an increasedrisk of having or developing immune tolerance or a decreased risk ofhaving or developing immune tolerance.

Similarly, the nucleic acid sequences of the present invention can beassociated with either an increased or decreased likelihood ofresponding to a particular treatment or antigen, or an increased ordecreased likelihood of developing immune tolerance to the particulartreatment or antigen.

In some embodiments, the nucleic acid sequences are SNPs. Such nucleicacid variation can be assayed in a number of different methods that arewell known to those of skill in the art, including PCR, RFLP,hybridization and direct sequencing.

Determination of Nucleic Acid Presence/Absence in IndividualsSusceptible to Immune Tolerance

One embodiment is to a method to determine nucleic acid sequences thatare present or absent in an individual who is susceptible to immunetolerance development, comprising screening for at least one nucleicacid sequence, as show in FIG. 1. In this method, the medicalpractitioner first administers an oral antigen to a patient to induceimmune tolerance (101).

Antigens suitable for use in the present invention include, but are notlimited to, synthetic or naturally derived proteins and peptides, andparticularly those which by themselves require high doses to induce oraltolerance; carbohydrates including, but not limited to, polysaccharidesand lipopolysaccharides; and antigens isolated from biological sourcessuch as, for example, those associated with or responsible for theinduction of auto-immune diseases, clinical (allergic)hypersensitivities, and allograft rejection and subunits or extractstherefrom; or any combination thereof.

Further, the antigens may be any associated with or responsible for theinduction of auto-immune diseases, clinical (allergic)hypersensitivities, and allograft rejection, and subunits or extractstherefrom; or recombinantly generated whole proteins, subunits orfragments thereof; or any combination thereof.

The antigen administered may include, but is not limited to, all of theantigens of Table 1, to treat the associated diseases. The associateddiseases or disorders listed in the Table 1 are meant to be examples andare in no way inclusive. In one specific embodiment, the at least oneantigen is a collagen selected from the group of: I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX,XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, and mixtures thereof.The collagen may also be a fragment thereof. For instance, the collagenmay be one or more fragments produced by CNBr cleavage of α1(I), whichyields eight CB fragments: CB0, CB1, CB2, CB4, CB5, CB8, CB3, CB7 andCB6. The collagen may be one or more fragments produced by cCNBrcleavage of α2(I), which yields six CB fragments: CB1, CB0, CB4, CB2,CB3 and CB5.

Further, in one embodiment, the disease is a sclerotic disease. Inanother embodiment, the disease is multiple sclerosis.

Table 1 shows multiple embodiments of the invention. For instance, inone embodiment, collagen is the antigen used to induce immune toleranceto the disease idiopathic pulmonary fibrosis. In another embodiment,α-enolase is an antigen used to induce immune tolerance for the diseaseasthma.

Furthermore, the individual dose size, number of doses, frequency ofdose administration, and mode of administration may vary. Determinationof such protocols can be accomplished by those skilled in the art. Asuitable single dose is a dose that is capable of altering a biologicalresponse in an animal when administered one or more times over asuitable time period (e.g., from minutes to days over weeks).Preferably, a dose comprises from about 1 ng of the antigen per kilogramof body weight (ng/kg) to about 1 gram of antigen per kilogram of bodyweight (gm/kg), more preferably 100 ng/kg to about 100milligrams/kilogram (mg/kg), and even more preferably from about 10microgram of the antigen per kilogram body weight (μg/kg) to about 10mg/kg.

Alternatively, the dose of antigen may not be determined upon the bodyweight of the patient. In this embodiment, the dose may be at least 1ng/day of antigen. Preferably, the dose ranges from 10 μg/day of antigento 5000 μg/day. In another embodiment, the dose ranges from 10 μg/day ofantigen to 500 μg/day. In another embodiment, the dose ranges from 30μg/day of antigen to 200 μg/day.

In one alternate embodiment, the dose of antigen is below the thresholdrequired for stimulation of an immune response. In another embodiment,the dose of antigen is above the threshold, thereby stimulating animmune response.

Modes of administration can include, but are not limited to,aerosolized, subcutaneous, rectally, intradermal, intravenous, nasal,oral, transdermal and intramuscular routes. In one preferred embodiment,the antigen is given orally. In another preferred embodiment, theantigen is given intraveneously or sublingually.

Furthermore, the antigen can be combined with other components such as apharmaceutically acceptable excipient and/or a carrier, prior toadministration to the individual. The other components will depend uponthe mode of administration, storage needs, and the like.

Examples of such excipients include water, saline, Ringer's solution,dextrose solution, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesameoil, ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity-enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers include,but are not limited to, phosphate buffer, bicarbonate buffer and Trisbuffer, while examples of preservatives include, but are not limited to,thimerosal, m- or o-cresol, formalin and benzyl alcohol.

Standard formulations can either be liquid injectables or solids thatcan be taken up in a suitable liquid as a suspension or solution forinjection. Carriers are typically compounds that increase the half-lifeof an antigen in the treated individual. Suitable carriers include, butare not limited to, polymeric controlled release vehicles, biodegradableimplants, liposomes, bacteria, viruses, oils, esters, and glycols.Preferred controlled release formulations are capable of slowlyreleasing the antigen of the present invention into an individual.Suitable controlled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, microparticles, bolus preparations, osmotic pumps,diffusion devices, liposomes, lipospheres, and transdermal deliverysystems. Other controlled release vehicles of the present inventioninclude liquids that, upon administration to an individual, form a solidor a gel in situ. Preferred controlled release vehicles arebiodegradable (i.e., bioerodible).

A biological sample, also referred to as a “sample”, is taken from theindividual for use in determining the individual's immune response(102). Such sample preparation components can be used to produce nucleicacid extracts (including DNA and/or RNA), proteins or membrane extractsfrom any bodily fluids (such as blood, serum, plasma, urine, saliva,phlegm, gastric juices, semen, tears, sweat, etc.), skin, hair, cells(especially nucleated cells), biopsies, buccal swabs or tissuespecimens. The frequency of taking samples, and its use, will vary basedon such factors as the scoring method, assay format, nature of thedetection method, and the specific tissues, cells or extracts used asthe test sample to be assayed. Methods of preparing nucleic acids,proteins, and cell extracts from the biological sample are well known inthe art and can be readily adapted to obtain a sample that is compatiblewith the system utilized.

From the biological sample, the individual immune system response to theantigen is determined, and the individuals are scored based upon theirresponse (103). The method of scoring is dependent upon thepractitioner, and includes any methods that separate patients based upontheir immune response to the antigen. For instance, the patient may bescored based on antigen specific and antigen non-specific assays.Antigen specific assays measure the response of T and B cells tospecific antigens, whereas antigen non-specific assays determine thephenotype of surface markers or functional state of cells for patternsassociated with a particular clinical status.

Specifically, the practitioner may use any means convenient to determinethe immune system response, including but not limited to, use of anenzyme-linked immunoabsorbent assay (ELISA), ELISA/ACT® LymphocyteResponse Assay (LRA), in vitro measurement of antibody production, mixedleukocyte reaction, cytotoxic T lymphocyte assay, flow cytometry,Western blots, limiting dilution assay, mass spectroscopy,immunoprecipitation and immunofluorescence.

For instance, in one embodiment, an ELISPOT assay is used to determinethe individual immune response. Typically, an ELISPOT assay includesincubating immune cells in plates coated with a capture antibody againsta cytokine of interest. Cytokines released by the cell membrane are thencaptured by the capture antibody during the incubation period. The cellsare removed and the bound cytokine is detected using a labeling system,such as for example, a labeled secondary antibody against a differentepitope of the same cytokine. This assay results in a cytokine footprintof a single cell.

In another embodiment, a transvivo DTH assay is used to determineindividual immune response. In this assay, cells from the individual areinjected into the footpads of immune-deficient mice together with theantigen. The index of reactivity of T cells to the antigens is thenmeasured by quantification of resultant swelling in the footpad.

In another embodiment, a tetramer assay is used. Here, the frequency ofT cells is measured by their binding to specific peptide-MHC complexesusing flow cytometry.

In an alternate embodiment, a CFSE assay measures the proliferation of Tcells by dilution of a CFSE dye in the dividing cells. This assay mayinclude use of flow cytometry.

In yet another embodiment, intracellular staining of the T cells can beused to determine the frequency of cytokine-producing T cells by, forexample, flow cytometry.

In yet another embodiment, the patients are scored based upon therespective levels and/or changes in IFN-γ production before, duringand/or after receiving the antigen. Alternatively, other cytokines maybe measured to determine individual scores. For instance, levels ofIL-10 in α1(I)- and α2(I)-stimulated PBMC culture supernatants and/orsIL-2R may be used to score a patient.

Other means to determine the immune system response may includecharacterization of the TCR repertoire. This assay may include use ofquantitative PCR, gel electrophoresis and DNA sequencing to determinethe proporation of T cells that use the Vβ chains to determine the CFR3length distribution along the Vβ gene product.

Another means to determine immune system response may include T cellresponses to polyclonal, non-antigen-specific stimulation. Here, thewhole blood is stimulated with phytohemagglutinin for a period of time,CD4+ T cells are then isolated, and the extent of early CD4+ T cellactivation is measured by the synthesis and accumulation ofintracellular ATP measured after cell lysis.

Other means to determine the immune system response may include, but isnot limited to, detection of the presence of nucleic acids. Severalmethods of detecting nucleic acids are available including PCR, LCR andhybridization techniques. Hybridization techniques involve detecting thehybridization of two or more nucleic acid molecules, where detection isachieved in a variety of ways, including labeling the nucleic acidmolecules and observing the signal generated from such a label.Hybridization techniques may include any of the following: Northern andSouthern blotting, cycling probe reaction, branched DNA, Invader™ Assay,and Hybrid Capture. Hybridization techniques may also be used toidentify a specific sequence of nucleic acid present in a sample byusing microarrays (or “bioarrays”) of known nucleic acid sequences toprobe a sample. Bioarray technologies generally use known singlestranded nucleic acid, where each unique short chain is attached in aspecific known location and then adding the sample nucleic acid andallowing sequences present in the sample to hybridize to the immobilizedstrands. Detection of this hybridization is then carried out bylabeling, typically end labeling, of the fragments of the sample to bedetected prior to the hybridization. Further, hybridization may bedetermined by use of a fluorescent in situ hybridization technique.

Furthermore, in one embodiment, a proteomics approach may be used. Here,protein microarrays or mass spectrometry may be used to determine thepresence and quantification of protein fragments present in anindividual's sample. In addition, by analyzing more than one protein,the practitioner can determine the immune response by the uniquepatterns of protein expression in the individual.

To score the patients based on immune system response, the practitionermay measure compare the immune response before, during and/or afterreceiving the antigen. The determination of when the immune response ismeasured, and what method is used, is based upon the practitioner needs.Further, it is contemplated that the practitioner may further take intoconsideration other physiological factors of the individual, such asother cytokines, percentage of T cell, NK cell, B cell, dendritic cell,monocyte, subpopulations, oxidative radicals, connective tissue growthfactor, nitric oxide, thymic morphology determined by imagingtechniques, patient height, nutritional status, weight, health, thymicfunction determined by immunologic assays, diet, gender, age, vitamin Alevels, zinc levels, and environmental considerations to assist inscoring the patient's immune response. Patients' genomic backgrounds,such as mutations in other genes or genome regions, races and genderdifferences, may also be considered.

Once the individual is scored based on antigen response, thepractitioner will analyze the genotype of the individuals to determinewhether an individual has or lacks one or more nucleic acid sequences,thereby altering levels or patterns of gene expression (104). Thegenotype is later used to correlate the presence or absence of the oneor more nucleic acids with the immune response score of step (103).

The practitioner may either examine specific known nucleic acids ofinterest or examine part or whole of the entire genome to look for thepresence or absence of differential or unique nucleic acids or nucleicacid patterns, and correlate the one or more nucleic acidspresence/absence with the individual's immune response score. Nucleicacids of particular interest include those known to affect theconcentration of mRNA or protein in a sample, nucleic acids known toaffect the kinetics of nucleic acid and/or protein expression, nucleicacids that affect the rate of nucleic acid and/or protein decomposition,and nucleic acids that affect protein stability profile, Km, or Vmax.

Further, the practitioner may either use part of the biological sampleof step (102) to analyze for the nucleic acid, or the practitioner maytake another biological sample from the individual for analysis. Thenucleic acid may be purified or isolated from the sample by any meansconvenient to the practitioner, but such isolation may not be necessaryin certain forms of detection.

In one preferred embodiment, the practitioner will analyze the genotypeof the individuals to determine which allele(s) is/are present at anygiven genetic region of interest by methods well known in the art. Theneighboring sequence can be used to design nucleic acid detectionreagents such as oligonucleotide probes, which may optionally beimplemented in a kit format.

Common genotyping methods include, but are not limited to, TaqManassays, molecular beacon assays, nucleic acid arrays, allele-specificprimer extension, allele-specific PCR, arrayed primer extension,homogeneous primer extension assays, primer extension with detection bymass spectrometry, pyrosequencing, multiplex primer extension sorted ongenetic arrays, ligation with rolling circle amplification, homogeneousligation, OLA, multiplex ligation reaction sorted on genetic arrays,restriction-fragment length polymorphism, single base extension-tagassays, and the Invader assay. Such methods may be used in combinationwith detection mechanisms such as, for example, luminescence orchemiluminescence detection, fluorescence detection, time-resolvedfluorescence detection, fluorescence resonance energy transfer,fluorescence polarization, mass spectrometry, and electrical detection.

Various methods for detecting polymorphisms include, but are not limitedto, methods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA, comparison of theelectrophoretic mobility of variant and wild type nucleic acidmolecules, and assaying the movement of polymorphic or wild-typefragments in polyacrylamide gels containing a gradient of denaturantusing denaturing gradient gel electrophoresis. Sequence variations atspecific locations can also be assessed by nuclease protection assayssuch as RNase and SI protection or chemical cleavage methods.

In one embodiment, genotyping is performed using the TaqMan assay, whichis also known as the 5′ nuclease assay. The TaqMan assay detects theaccumulation of a specific amplified product during PCR. The TaqManassay utilizes an oligonucleotide probe labeled with a fluorescentreporter dye and a quencher dye. The reporter dye is excited byirradiation at an appropriate wavelength, it transfers energy to thequencher dye in the same probe via a process called fluorescenceresonance energy transfer (FRET). When attached to the probe, theexcited reporter dye does not emit a signal. The proximity of thequencher dye to the reporter dye in the intact probe maintains a reducedfluorescence for the reporter. The reporter dye and quencher dye may beat the 5′ most and the 3′ most ends, respectively, or vice versa.Alternatively, the reporter dye may be at the 5′ or 3′ most end whilethe quencher dye is attached to an internal nucleotide, or vice versa.In yet another embodiment, both the reporter and the quencher may beattached to internal nucleotides at a distance from each other such thatfluorescence of the reporter is reduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

Preferred TaqMan primer and probe sequences can readily be determinedusing the SNP and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the nucleic acids of the presentinvention are useful in diagnostic assays for stenosis and relatedpathologies, and can be readily incorporated into a kit format. Thepresent invention also includes modifications of the Taqman assay wellknown in the art such as the use of Molecular Beacon probes and othervariant formats.

Another method for genotyping the nucleic acids of the present inventionis the use of two oligonucleotide probes in an OLA. In this method, oneprobe hybridizes to a segment of a target nucleic acid with its 3′ mostend aligned with the nucleic acid site. A second probe hybridizes to anadjacent segment of the target nucleic acid molecule directly 3′ to thefirst probe. The two juxtaposed probes hybridize to the target nucleicacid molecule, and are ligated in the presence of a linking agent suchas a ligase if there is perfect complementarity between the 3′ mostnucleotide of the first probe with the nucleic acid site. If there is amismatch, efficient ligation cannot occur. After the reaction, theligated probes are separated from the target nucleic acid molecule, anddetected as indicators of the presence of a nucleic acid sequence. OLAmay also be used for performing nucleic acid detection using universalarrays, wherein a zipcode sequence can be introduced into one of thehybridization probes, and the resulting product, or amplified product,hybridized to a universal zip code array. Alternatively OLA may be usedwhere zipcodes are incorporated into OLA probes, and amplified PCRproducts are determined by electrophoretic or universal zipcode arrayreadout.

Alternatively one may use SNPlex methods and software for multiplexedSNP detection using OLA followed by PCR, wherein zipcodes areincorporated into OLA probes, and amplified PCR products are hybridizedwith a zipchute reagent, and the identity of the SNP determined fromelectrophoretic readout of the zipchute. In some embodiments, OLA iscarried out prior to PCR (or another method of nucleic acidamplification). In other embodiments, PCR (or another method of nucleicacid amplification) is carried out prior to OLA.

Another method for genotyping is based on mass spectrometry. Massspectrometry takes advantage of the unique mass of each of the fournucleotides of DNA. Nucleic acids can be unambiguously genotyped by massspectrometry by measuring the differences in the mass of nucleic acidshaving alternative nucleic acid alleles. MALDI-TOF (Matrix AssistedLaser Desorption Ionization—Time of Flight) mass spectrometry technologyis preferred for extremely precise determinations of molecular mass,such as for SNPs. Numerous approaches to genotype analysis have beendeveloped based on mass spectrometry. Preferred mass spectrometry-basedmethods of nucleic acid genotyping include primer extension assays,which can also be utilized in combination with other approaches, such astraditional gel-based formats and microarrays.

Typically, the primer extension assay involves designing and annealing aprimer to a template PCR amplicon upstream (5′) from a target nucleicacid position. A mix of dideoxynucleotide triphosphates (ddNTPs) and/ordeoxynucleotide triphosphates (dNTPs) are added to a reaction mixturecontaining template. For example, in some embodiments this is aSNP-containing nucleic acid molecule which has typically been amplified,such as by PCR. Primer and DNA polymerase may further be added.Extension of the primer terminates at the first position in the templatewhere a nucleotide complementary to one of the ddNTPs in the mix occurs.The primer can be either immediately adjacent (i.e., the nucleotide atthe 3′ end of the primer hybridizes to the nucleotide next to the targetSNP site) or two or more nucleotides removed from the nucleic acidposition. If the primer is several nucleotides removed from the targetnucleic acid position, the only limitation is that the template sequencebetween the 3′ end of the primer and the nucleic acid position cannotcontain a nucleotide of the same type as the one to be detected, or thiswill cause premature termination of the extension primer.

Alternatively, if all four ddNTPs alone, with no dNTPs, are added to thereaction mixture, the primer will always be extended by only onenucleotide, corresponding to the target SNP position. In this instance,primers are designed to bind one nucleotide upstream from the SNPposition (i.e., the nucleotide at the 3′ end of the primer hybridizes tothe nucleotide that is immediately adjacent to the target SNP site onthe 5′ side of the target SNP site). Extension by only one nucleotide ispreferable, as it minimizes the overall mass of the extended primer,thereby increasing the resolution of mass differences betweenalternative SNP nucleotides. Furthermore, mass-tagged ddNTPs can beemployed in the primer extension reactions in place of unmodifiedddNTPs. This increases the mass difference between primers extended withthese ddNTPs, thereby providing increased sensitivity and accuracy, andis particularly useful for typing heterozygous base positions.Mass-tagging also alleviates the need for intensive sample-preparationprocedures and decreases the necessary resolving power of the massspectrometer.

The extended primers can then be purified and analyzed by MALDI-TOF massspectrometry to determine the identity of the nucleotide present at thetarget SNP position. In one method of analysis, the products from theprimer extension reaction are combined with light absorbing crystalsthat form a matrix. The matrix is then hit with an energy source such asa laser to ionize and desorb the nucleic acid molecules into thegas-phase. The ionized molecules are then ejected into a flight tube andaccelerated down the tube towards a detector. The time between theionization event, such as a laser pulse, and collision of the moleculewith the detector is the time of flight of that molecule. The time offlight is precisely correlated with the mass-to-charge ratio (m/z) ofthe ionized molecule. Ions with smaller m/z travel down the tube fasterthan ions with larger m/z and therefore the lighter ions reach thedetector before the heavier ions. The time-of-flight is then convertedinto a corresponding, and highly precise, m/z. In this manner, SNPs canbe identified based on the slight differences in mass, and thecorresponding time of flight differences, inherent in nucleic acidmolecules having different nucleotides at a single base position.

Nucleic acids can also be scored by direct DNA sequencing. A variety ofautomated sequencing procedures can be used, including sequencing bymass spectrometry. The nucleic acid sequences of the present inventionenable one of ordinary skill in the art to readily design sequencingprimers for such automated sequencing procedures. Commercialinstrumentation, such as the Applied Biosystems 377, 3100, 3700, 3730,and 3730×1 DNA Analyzers (Foster City, Calif.), is commonly used in theart for automated sequencing. Nucleic acid sequences can also bedetermined by employing a high throughput mutation screening system,such as the SpectruMedix system.

Other methods that can be used to genotype the nucleic acids of thepresent invention include single-strand conformational polymorphism(SSCP), and denaturing gradient gel electrophoresis (DGGE). SSCPidentifies base differences by alteration in electrophoretic migrationof single stranded PCR products. Single-stranded PCR products can begenerated by heating or otherwise denaturing double stranded PCRproducts. Single-stranded nucleic acids may refold or form secondarystructures that are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts are related to base-sequence differences at nucleic acidpositions. DGGE differentiates nucleic acid alleles based on thedifferent sequence-dependent stabilities and melting properties inherentin polymorphic DNA and the corresponding differences in electrophoreticmigration patterns in a denaturing gradient gel.

Sequence-specific ribozymes can also be used to score nucleic acids, inparticular SNPs, based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. Thus, for example, if the SNP affects a restrictionenzyme cleavage site, the SNP can be identified by alterations inrestriction enzyme digestion patterns, and the corresponding changes innucleic acid fragment lengths determined by gel electrophoresis

Genotyping can include the steps of, for example, collecting abiological sample from a human subject (e.g., sample of tissues, cells,fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA,mRNA or both) from the cells of the sample, contacting the nucleic acidswith one or more primers which specifically hybridize to a region of theisolated nucleic acid containing a target nucleic acid region ofinterest under conditions such that hybridization and amplification ofthe target nucleic acid region occurs, and determining the nucleotidepresent at the nucleic acid position of interest, or, in some assays,detecting the presence or absence of an amplification product (assayscan be designed so that hybridization and/or amplification will onlyoccur if a particular nucleic acid sequence allele is present orabsent). In some assays, the size of the amplification product isdetected and compared to the length of a control sample; for example,deletions and insertions can be detected by a change in size of theamplified product compared to a normal genotype.

Furthermore, the nucleic acid, or in particular the SNP, found may thenbe compared to the nucleic acids of other individuals whom have alsoreceived the antigen to induce an immune response. Methods of comparingthe identity of two or more sequences may be performed by any reasonablemeans, including programs available in the Wisconsin Sequence AnalysisPackage version 9.1 (Genetics Computer Group, Madison, Wis., USA). Otherprograms such as BESTFIT may be used to find the “local homology”algorithm of Smith and Waterman and finds the best single region ofsimilarity between two sequences. Further, programs such as GAP may beused, which aligns two sequences finding a “maximum similarity.”Preferably, % identities and similarities are determined when the twosequences being compared are optimally aligned. Other programs fordetermining identity and/or similarity between sequences are also knownin the art, for instance the BLAST family of programs, available fromthe National Center for Biotechnology Information (NCB), Bethesda, Md.,USA) and FASTA, available as part of the Wisconsin Sequence AnalysisPackage.

Once the presence or absence or pattern of nucleic acids present in anindividual is determined, the immune response score is associated withthe nucleic acid results. The practitioner may then determine if one ormore nucleic acids, preferably SNPs, are associated with individuals whodid or did not respond to the antigen with altered immune tolerance.

It is contemplated that the mechanisms of induction of immune tolerancemay vary with different diseases and disorders. For example, the SNPinvolved with induction of systemic lupus erthematosus may be differentfrom the SNPs involved with other diseases, such as autoimmuneencephalopathy. As such, the practitioner may repeat the process of FIG.1 for each disease and disorder that the pracititioner wishes to induceimmune tolerance to.

Determination of Treatment Based Upon an Individual's Genotyping

The method of FIG. 1 is particularly beneficial because it allows thepractitioner to determine what nucleic acid sequences, including SNPs,may be involved with induction of immune tolerance. More importantly,once the method of FIG. 1 is complete, the practitioner may simply testa new individual for the nucleic acid sequence or SNP of interest, asoutlined in FIG. 2.

First, the practitioner categorizes or diagnoses the patient as having adisease or disorder (201). The categorization or diagnosis may be basedon present or past symptoms, medical history, family history, tests suchas assays or medical scans, and the like.

Once the practitioner has diagnosed the patient, the practitioner takesa biological sample from the patient (202). The sample may be in anyform convenient for the practitioner and patient, including but notlimited to, whole blood, blood plasma, urine, tears, semen, saliva,buccal mucosa, interstitial fluid, lymph fluid, meningeal fluid,amniotic fluid, glandular fluid, sputum, feces, perspiration, mucous,vaginal secretion, cerebrospinal fluid, hair, skin, fecal material,wound exudate, wound homogenate, and wound fluid.

The individual's nucleic acid is then isolated from the sample (203).The isolation may occur by any means convenient to the practitioner. Forinstance, the isolation may occur by first lysing the cell usingdetergents, enzymatic digestion or physical disruption. Thecontaminating material is then removed from the nucleic acids by use of,for example, enzymatic digestion, organic solvent extraction, orchromatographic methods. The individual's nucleic acid may be purifiedand/or concentrated by any means, including precipitation with alcohol,centrifugation and/or dialysis.

The individual's nucleic acid is then assayed for presence or absence ofone or more predetermined nucleic acids of interest (204). The one ormore predetermined nucleic acids of interest may be any nucleic acid thepractitioner believes may be related to immune tolerance to any diseaseor disorder. Exemplary examples of appropriate diseases or disorders arelisted in Table 1.

The determination of whether one or more predetermined nucleic acids ofinterest are presence or absence, may be done by any mean (205).

In one embodiment, the predetermined nucleic acid of interest is anapproximately 50 KB non gene region on chromosome 12, located about 55.5Mbp from the proximal end of chromosome 12 between 66,603,791 and66,603,991 bps, where the nucleic acid of interest is eitherTTTTTTTTTTGTACCTAGTTCTATGGTTACCTT (SEQ ID NO. 1) orTTTTTTTTTTGTACCTGGTTCTATGGTTACCTT (SEQ ID NO. 2). The A/G is thepolymorphic site. Thus, AA represents AA homozygous, while AB representsA/G heterozygous and BB represents GG genotype, A→G represents thepolymorphism site.

Alternatively, the predetermined nucleic acid of interest may includepart of the approximately 265143 by at 5′ side of SNP_A-1515737.Alternatively, the predetermined nucleic acid of interest may includepart of the approximately 231513 by at 3′ side of SNP-1515737.Alternatively, the predetermined nucleic acid of interest includes partof all of SEQ. ID. 3 or 4.

In another embodiment, the predetermined nucleic acid of interest islocated between 66507155-66507464 on chromosome 12, within the proximityof the marker D1251503.

In another embodiment, the predetermined nucleic acid of interest islocated within the proximity of D1251676, i.e., part or all of thepredetermined nucleic acid of interest is located between66499298-66499423 on chromosome 12.

In another embodiment, the predetermined nucleic acid of interest islocated within the proximity of D12S335, i.e., part or all of thepredetermined nucleic acid of interest is located between66415802-66416056 on chromosome 12.

In another embodiment, the predetermined nucleic acid of interest islocated within the proximity of D12S102, i.e., part or all of thepredetermined nucleic acid of interest is located between66781046-66781298 on chromosome 12.

In another embodiment, the predetermined nucleic acid of interest islocated within the proximity of D12S1506, i.e., part or all of thepredetermined nucleic acid of interest is located between66785380-66785614 on chromosome 12.

In another embodiment, the predetermined nucleic acid of interest isSNP_A-1508498 (TSC51977), with the polymorphism of C or T. In anotherembodiment, the predetermined nucleic acid of interest is SNP_A-1512645(TSC1720860) with the polymorphism of C or T. In another embodiment, thepredetermined nucleic acid of interest is SNP_A-1512719 (TSC1720861)with the polymorphism of C or T. In another embodiment, thepredetermined nucleic acid of interest is SNP_A-1515330 (TSC1244733)with the polymorphism of C or T. In another embodiment, thepredetermined nucleic acid of interest is SNP_A-1518829 (TSC51583) withthe polymorphism of A or G. In another embodiment, the predeterminednucleic acid of interest is SNP_A-1518878 (TSC51584) with thepolymorphism of C or G. Further, in one specific embodiment, thepredetermined nucleic acid of interest is located within 30 kbp ofSNP_A-1515737. These other markers and transcripts can also be used todetermine a patient's ability to have tolerance, especially oraltolerance, induced.

In another embodiment, the predetermined nucleic acid of interest isD12S1503 (SEQ. ID. NO. 5). In another embodiment, the predeterminednucleic acid of interest is D1251676 (SEQ. ID. NO. 6). In anotherembodiment, the predetermined nucleic acid of interest is D125335 (SEQ.ID. NO. 7). In another embodiment, the predetermined nucleic acid ofinterest is D125102 (SEQ. ID. NO. 8).

In another embodiment, the predetermined nucleic acid of interest isSNP_A-1508498 with a polymorphism of C or T. (SEQ. ID. NO. 9 and 10). Inanother embodiment, the predetermined nucleic acid of interest isSNP_A-1512645 with a polymorphism of C or T. (SEQ. ID. NO. 11 or 12). Inanother embodiment, the predetermined nucleic acid of interest isSNP_A-1512719 with a polymorphism of C or T. (SEQ. ID. NO. 13 or 14). Inanother embodiment, the predetermined nucleic acid of interest isSNP_A-1515330 with a polymorphism of C or T. (SEQ. ID. NO. 15 or 16). Inanother embodiment, the predetermined nucleic acid of interest isSNP_A-1518829 with a polymorphism of A or G. (SEQ. ID. NO. 17 or 18). Inanother embodiment, the predetermined nucleic acid of interest isSNP_A-1518878 with a polymorphism of C or G. (SEQ. ID. NO. 19 or 20).The predetermined nucleic acid of interest may also be any marker orgene between 63.3 mbp and 69.4 mbp on human chromosome 12. See, e.g.,FIG. 15.

In one embodiment, the practitioner uses visual confirmation of thepresence or absence of particular variants of the nucleic acid.

In another embodiment, the method provides a computer based system withone or more algorithms to determine the presence and/or absence of oneor more predetermined nucleic acids of interest and, if present,quantifies the amount of one or more predetermined nucleic acids ofinterest present in the individual's nucleic acid.

In one preferred embodiment, the predetermined nucleic acids of interestare one or more SNPs. Further, the computer based system may compriseinformation about observed SNP alleles, alternative codons, populations,allele frequencies, SNP types, and/or affected proteins and the like.

The computer based system includes at least one of the following:hardware means, software means, and data storage means used to analyzeany information of the present invention. The minimum hardware means ofthe computer-based systems of the present invention typically comprisesa central processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. Such a system can be changed into a system of thepresent invention by utilizing information provided on the CD-R, or asubset thereof, without any experimentation.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein information and thenecessary hardware means and software means for supporting andimplementing a search means. The search means of the computer-basedsystem includes one or more software programs or algorithms that areimplemented on the computer-based system to identify or analyze nucleicacid sequences, including SNPs in a target sequence, based on nucleicacid information stored within the data storage means. Search means canbe used to determine the presence or absence of a nucleic acid sequence,and/or which nucleotide is present at a particular SNP position in anucleic acid sequence.

In one application of this embodiment, the practitioner may provide thecomputer-based system with information regarding nucleic acids ofinterests on a computer readable medium. Computer readable medium is anymedium that can be read and accessed directly by a computer, includingbut are not limited to: magnetic storage media, such as floppy discs,hard disc storage medium, and magnetic tape; optical storage media suchas CD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleic acidsequence information of the present invention on computer readablemedium. For example, the sequence information can be represented in aword processing text file, formatted in commercially-available softwaresuch as WordPerfect and Microsoft Word, represented in the form of anASCII file, or stored in a database application, such as OB2, Sybase,Oracle, or the like. A skilled artisan can readily adapt any number ofdata processor structuring formats (e.g., text file or database) inorder to obtain computer readable medium having recorded thereon thenucleic acid sequence information of the present invention.

By providing the nucleic acid sequences, including SNPs, of the presentinvention in computer readable form, a practitioner can routinely accessthe nucleic acid sequence information for a variety of purposes.Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.Examples of publicly available computer software include BLAST and BLAZEsearch algorithms.

The present invention further provides systems, particularlycomputer-based systems, which contain the nucleic acid sequenceinformation described herein. Such systems may be designed to storeand/or analyze information on, for example, a large number of SNPpositions, or information on genotypes, including SNP genotypes, from alarge number of individuals. The nucleic acid sequence information ofthe present invention represents a valuable information source. Thenucleic acid sequence information of the present inventionstored/analyzed in a computer-based system may be used for suchcomputer-intensive applications as determining or analyzing nucleic acidallele frequencies in a population, mapping disease genes,genotype-phenotype association studies, grouping SNPs into haplotypes,correlating SNP haplotypes with response to particular drugs, or forvarious other bioinformatic, pharmacogenomic, drug development, or humanidentification/forensic applications.

A treatment for the individual is then formulated based on the presenceand/or absence of one or more predetermined nucleic acids of interest(206). The treatment may include anything within the means of thepractitioner, including a determination of likelihood of inducing immunetolerance can be induced, if so then the type of antigen, dose, methodof administration, regiment of treatment, and the like.

The inventors have also discovered that non-steroidal anti-inflammatorydrugs (NSAIDS) can interfere with the generation of immune tolerance.Thus, in one alternate embodiment, the practitioner can modulate thecreation of immune tolerance by either having the individual desiringtreatment stop using NSAIDS or administering pharmaceuticals to reversethe NSAID inhibition of immune tolerance, such as, for example,misoprostol.

Further, in one preferred embodiment, the methods of the instantinvention may be used to treat idiopathic pulmonary fibrosis (“IPF”).IPF is a lethal, chronic, progressive, interstitial lung disease inwhich normal lung tissue is gradually replaced by fibrotic tissue, or anabnormal and excessive amount of fibrotic tissue is deposited in thepulmonary interstitium. This may be described as a scarring of the lung.About 60% of IPF patients have an antigen-specific autoimmune reactionto Type V collagen. Without wishing to be bound to a particularmechanism or theory, it is believed that the immune systems of suchpatients attack the Type V collagen of the lungs, thereby causingfibrosis.

IPF patients having an autoimmune reaction to Type V collagen wouldbenefit if the immune response attacking the Type V collagen of thelungs could be halted or lessened. This immune response could be haltedor lessened by induction of immune tolerance to Type V collagen usingthe methods of the present invention. Tolerance could be induced byrepeated administration of collagen antigens, preferably Type V collagenantigens, according to the methods of the present invention.

Administration of collagen or collagen antigens, preferably Type Vcollagen or antigens thereof, will be most effective in treating IPFpatients who have an antigen-specific autoimmune reaction to Type Vcollagen. Thus, a test for identifying such patients is desirable.

Using the methods of the present invention, an association study may beperformed to determine whether IPF patients having an antigen-specificautoimmune reaction to Type V collagen carry one or more SNPs—or othernucleic acid sequences linked in some manner to the SNPs—that is notfound in IPF patients who do not have an antigen-specific autoimmunereaction to Type V collagen. The presence of the one or more SNPs orlinked nucleic acid sequences associated with an antigen-specificautoimmune reaction to Type V collagen can then be used to identifypatients most likely to benefit from administration of collagen orcollagen antigens, preferably Type V collagen or antigens thereof.Autoimmune reactions to Type V collagen are also believed to contributeto rejection of lung transplants. Accordingly, SNP(s) and/or linkednucleic acid sequences found to be associated with an antigen-specificautoimmune reaction to Type V collagen may also be used to assess apatient's propensity to successfully undergo lung or other transplants.

Use of Individual Genotype to Determine Whether to Induce ImmuneTolerance

The invention may also be used by the practitioner to determine theregiment of treatment for the individual. That is, based onindividual-specific information, the invention may recommend to thepractitioner treatment regiments, such as whether to induce immunetolerance, type of antigen, dose, method of administration, regiment oftreatment, and the like. The recommendation of treatment regiments maynot only be a function of the disease or disorder the individual has,but also a function of the individual characteristics.

In this embodiment, the method of which is located in FIG. 3, theindividual characteristics are first placed into one or more databases(301). The individual characteristic information may be received by anymeans, including using an integrated consultation process with one ormore practitioners, a questionnaire filled out by the individual, anelectronic database, a diagnostic or an expert panel measurement tool,client summary report, and an outcomes measurement report. Further, theindividual characteristic information may be received by one or more ofa network, oral communication, visual communication, writtencommunication, physical data carrier, and/or any other means capable ofconveying information.

Individual characteristics may include, but are not limited to, age,sex, height, weight, individual medical history, family medical history,ethnicity, allergy information, lifestyle information, and the like.

The individual's nucleic acids are then collected and analyzed (302).The method of collecting the individual nucleic acid may be anycontemplated by the practitioner, including those mentioned within theinstant specification.

The presence or absence of one or more predetermined nucleic acids ofinterest is then determined (303). Here, the determination which nucleicacids would be examined, i.e., the predetermined nucleic acids ofinterest, may be determined either individually by the practitioner.Alternatively, the determination includes accessing a databasecontaining information reflecting the relationship between the currentdisease or diagnosis of the individual, or what the practitionerbelieves the current disease or diagnosis to be, and the nucleic acidsof interest associated with the disease or diagnosis.

In some embodiments, the one or more databases of the current inventionare local to the practitioner's location. In other embodiments, the oneor more databases are remote, dynamic databases. Generally, a dynamicdatabase is one in which the data within may be easily changed orupdated. For instance, one can use a software system to accessinformation from a dynamic database via a network and upload informationfrom the database to the software system. If the information stored inthe database changes, the software system connected to the database willalso change accordingly and automatically without human intervention.The software system may update the individual's information in thedynamic database on any time bases, including, but not limited to, anevent driven, minute-by-minute, hourly, daily or weekly basis.

Based upon the results of step (303), i.e., the presence or absence ofone or more predetermined nucleic acids of interest, the treatment ofthe individual may be determined. In one embodiment, the practitionerrelies upon his expertise in the medical field to determine thetreatment regiment. In another embodiment, a software system stored inone or more databases recommends a treatment regiment. The recommendedtreatment regiment may include, but is not limited to, whether to induceimmune tolerance, type of antigen, dose, method of administration,regiment of treatment, over the counter or prescription medication,lifestyle changes, and the like.

The recommendation of treatment regiments may not only be a function ofthe disease or disorder the individual has, but also a function of theindividual characteristics. As such, the one or more databases may allowa software system to input more information about the individual, thedisease or disorder to be treated, the outcome of the treatment, andother compatible information. It is contemplated that the informationfor multiple individuals may be pooled together, thus as the amount ofdata for one or more parameters increases, the algorithm in the softwaresystem becomes more robust and accurate at calculating whattreatment/procedure best suits the individual's set of criteria.Further, the database may further include a warehouse of “BestPractices,” that is, specific treatment protocols judged optimal by apanel of medical experts.

These embodiments allow the practitioner to better tailor theindividual's treatment and avoid unnecessary cost and time ofunnecessary treatments.

Determination of Nucleic Acids Associated with Immune Tolerance

The determination of which nucleic acids are the predetermined nucleicacids of interest may be determined by the method of FIG. 4. First,information on multiple individuals with a disease or disorder iscollected and saved in one or more databases (401). The informationincludes at least the disease or disorder of the individuals, theability of the individual to develop immune tolerance afteradministration of antigens, and the nucleic acid information of theindividual. Preferably, the nucleic acid information is the presence orabsence of one or more SNPs.

The presence of absence of one or more SNPs and the ability of theindividual to develop immune tolerance is compared to determine theincreased (or decreased) occurrence of the nucleic acid in a specificdisease or disorder condition (402). The comparison of the SNPs andability to develop immune tolerance may be accomplished by using amathematical algorithm.

Once a statistically significant association is established between oneor more SNPs and ability to induce immune tolerance, then the regionaround the SNP can optionally be thoroughly screened to identify thecausative genetic locus/sequence(s) (e.g., causative SNP/mutation, gene,regulatory, region, etc.) that influences the ability to induce immunetolerance.

In addition, an association study of a SNP and a specific disease ordisorder may be performed, to determine the presence or frequency of theSNP allele in biological samples from individuals with the disorder ordisease of interest and comparing the information to that of controls(i.e., individuals who do not have the disorder; controls may be alsoreferred to as “healthy” or “normal” individuals) who are preferably ofsimilar age and race. The patients and controls should be as alike aspossible in physical characteristics, and a pool of individuals withwell-characterized phenotypes is extremely desirable. Further,association studies may also be conducted within the general populationand are not limited to studies performed on related individuals inaffected families (linkage studies).

The information on the one or more SNPs is then stored in one or moredatabases, along with information regarding its presence or absence inindividuals with the ability to induce immune tolerance (403).

The nucleic acid of individuals who desire treatment for a disease ordisorder is then collected and analyzed to find if the individual hasthe one or more SNPs that are statistically significant associated withthe ability of other individuals to induce immune tolerance (404). Themedical practitioner alone, or with the assistance of a software systemcomprising one or more databases, may then determine what medicalregiments are feasible, and the parameters of those medical treatments.

Determination of Altered Polypeptide Presentation in IndividualsSusceptible to Immune Tolerance

In another embodiment, a method is first used to determiningpolypeptides that are present or absent in an individual who issusceptible to immune tolerance development, comprising screening for atleast one polypeptide, as show in FIG. 5. In this method, the medicalpractitioner first administers an oral antigen to a patient to induceimmune tolerance (501).

Antigens suitable for use in the present invention include thosepreviously discussed, and may include any associated with or responsiblefor the induction of auto-immune diseases, clinical (allergic)hypersensitivities, and allograft rejection, and subunits or extractstherefrom; or recombinantly generated whole proteins, subunits orfragments thereof; or any combination thereof. Furthermore, the antigenmay include, but is not limited to, all of the antigens of Table 1, totreat the associated diseases. The antigen may further be combined withother components such as a pharmaceutically acceptable excipient and/ora carrier, prior to administration to the individual.

The individual dose size, number of doses, frequency of doseadministration, and mode of administration may vary be determined bythose skilled in the art. Suitable doses of antigen are those previouslydiscussed. Further, the modes of administration can include, but are notlimited to, aerosolized, subcutaneous, rectally, intradermal,intravenous, nasal, oral, transdermal and intramuscular routes.

A biological sample is taken from the individual for use in determiningthe individual's immune response (502), whereby the individual immunesystem response to the antigen is scored based upon their response(503).

The method to determine the individual immune response may include anymethod previously discussed, including but not limited to, use of anenzyme-linked immunoabsorbent assay (ELISA), ELISA/ACT® LymphocyteResponse Assay (LRA), in vitro measurement of antibody production, mixedleukocyte reaction, cytotoxic T lymphocyte assay, flow cytometry,Western blots, limiting dilution assay, mass spectroscopy,immunoprecipitation, immunofluorescence, ELISPOT, transvivo DTH assay,tetramer assay, CFSE assay, characterization of the TCR repertoire,measuring T cell responses to polyclonal, non-antigen-specificstimulation, detection of the presence of nucleic acids including PCR,LCR, hybridization techniques and proteomics. In addition, in onespecific embodiment, the patients are scored based upon the respectivelevels and/or changes of cytokine production, including IL-17, IL-2 orIFN-γ production before, during and/or after receiving the antigen.Further, levels of T regulatory cells such as Tr1 cells or CD4⁺, CD25⁺,FoxP3⁺, cells can be used to score patient response. Alternatively,other cytokines may be measured to determine individual scores. Forinstance, levels of IL-10, IL-4, IL-5 or TGF-β1, 2 or 3 in α1(I)- andα2(I)-stimulated PBMC culture supernatants and/or sIL-2R may be used toscore a patient.

The method of scoring is dependent upon the practitioner, and includesany methods that separate patients based upon their immune response tothe antigen. To score the patients based on immune system response, thepractitioner may measure compare the immune response before, duringand/or after receiving the antigen. The determination of when the immuneresponse is measured, and what method is used, is based upon thepractitioner needs. Further, it is contemplated that the practitionermay further take into consideration other physiological factors of theindividual, such as other cytokines, oxidative radicals, connectivetissue growth factor, nitric oxide, patient height, weight, health,diet, and environmental considerations to assist in scoring thepatient's immune response.

Once the individual is scored based on antigen response, thepractitioner will analyze the polypeptides of the individuals todetermine whether an individual has one or more polypeptides or variantpolypeptides (504).

The practitioner may either examine specific known polypeptides ofinterest or examine part or whole of the proteins present in the sampleto look for the presence or absence of differential or uniquepolypeptides or polypeptide patterns, and correlate the polypeptidespresence/absence with the individual's immune response score.

Polypeptides may be detected by various methods known to one skilled inthe art. Before polypeptide detection, the polypeptides within theindividual's biological sample may be purified to substantial purity bystandard techniques, including but not limited to, selectiveprecipitation with such substances as ammonium sulfate, cold ethanolprecipitation, ultrafiltration, column chromatography,immunopurification methods, and the like.

The polypeptides may be detected by use of any method convenient to thepractitioner, including, but not limited to sandwich assays andcompetition or displacement assays. Typically, a sandwich orcompetition/displacement assays includes a “capture agent” thatspecifically bind to and often immobilize the analyte (in this case oneor more polypeptides in the sample). The capture agent is a moiety thatspecifically binds to the analyte.

The presence or absence of the polypeptide may be determined by anymeans convenient to the practitioner, including electrochemical means oruse of labels. A label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. The label may be coupled directly orindirectly to the desired component of the assay according to methodswell known in the art. The antibody may be produced by any of a numberof means well known to those of skill in the art and as described above.Further, the label may be on a third moiety that binds to the captureagent/analyte complex, or secondarily binds to a moiety distinct for thecapture agent/analyte complex.

Other techniques that may be used to detect and/or quantify thepolypeptide includes western blot (immunoblot) analysis, liposomeimmunoassays (LIA), proteomics such as protein microarrays or massspectrometry.

The amino acid sequence of the polypeptides of interest found in theindividual's sample may be determined, and compared to one or moredatabases containing polypeptides amino acid sequences that arestatistically significant associated with the ability of otherindividuals to induce immune tolerance (404). The medical practitioneralone, or with the assistance of a software system comprising one ormore databases, may then determine what medical regiments are feasible,and the parameters of those medical treatments.

It is recognized that in any of the embodiments described herein, thedisease or disorder may be linked to race, ethnicity, or sex. Forexample, one study found women comprise 78% of diagnosed autoimmunediseases. Further, systemic sclerosis has a sex and race specificprevalence, where women are more likely than men to have the disorder,and African Americans are more likely then Caucasians to have thedisorder. As such, the presence or absence of the nucleic acid ofinterest may be tied to a hormone, the X chromosome, or to enzymes,receptors, or other compounds that have different levels between thesexes and or races.

SNP Detection Kits and Systems

Based on the SNP and associated sequence information disclosed herein,detection reagents can be developed and used to assay any SNP of thepresent invention individually or in combination, and such detectionreagents can be readily incorporated into one of the established kit orsystem formats which are well known in the art. Kits used in the contextof SNP detection reagents, are intended to refer to such things ascombinations of multiple SNP detection reagents, or one or more SNPdetection reagents in combination with one or more other types ofelements or components (e.g., other types of biochemical reagents,containers, packages such as packaging intended for commercial sale,substrates to which SNP detection reagents are attached, electronichardware components, etc.). Accordingly, the present invention furtherprovides SNP detection kits and systems, including but not limited to,packaged probe and primer sets (e.g., TaqMan probe/primer sets),arrays/microarrays of nucleic acid molecules, and beads that contain oneor more probes, primers, or other detection reagents for detecting oneor more SNPs of the present invention. The kits/systems can optionallyinclude various electronic hardware components; for example, arrays(“DNA chips”) and microfluidic systems (“lab-on-a-chip” systems)provided by various manufacturers typically comprise hardwarecomponents.

Other kits/systems (e.g., probe/primer sets) may not include electronichardware components, but may be comprised of, for example, one or moreSNP detection reagents (along with, optionally, other biochemicalreagents) packaged in one or more containers.

In some embodiments, a SNP detection kit typically contains one or moredetection reagents and other components (e.g., a buffer, enzymes such asDNA polymerases or ligases, chain extension nucleotides such asdeoxynucleotide triphosphates, and in the case of Sanger-type DNAsequencing reactions, chain terminating nucleotides, positive controlsequences, negative control sequences, and the like) necessary to carryout an assay or reaction, such as amplification and/or detection of aSNP-containing nucleic acid molecule. A kit may further contain meansfor determining the amount of a target nucleic acid, and means forcomparing the amount with a standard, and can comprise instructions forusing the kit to detect the SNP-containing nucleic acid molecule ofinterest. In one embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out one or more assays todetect one or more SNPs disclosed herein. In a preferred embodiment ofthe present invention, SNP detection kits/systems are in the form ofnucleic acid arrays, or compartmentalized kits, includingmicrofluidic/lab-on-a-chip systems.

SNP detection kits/systems may contain, for example, one or more probes,or pairs of probes, that hybridize to a nucleic acid molecule at or neareach target SNP position. Multiple pairs of allele-specific probes maybe included in the kit/system to simultaneously assay large numbers ofSNPs, at least one of which is a SNP of the present invention. In somekits/systems, the allele-specific probes are immobilized to a substratesuch as an array or bead. For example, the same substrate can compriseallele-specific probes for detecting at least 1; 10; 100; 1000; 10,000;100,000 (or any other number in-between) or substantially all of theSNPs shown in Table 1 and/or Table 2.

The terms “arrays”, “microarrays”, and “DNA chips” are used hereininterchangeably to refer to an array of distinct polynucleotides affixedto a substrate, such as glass, plastic, paper, nylon or other type ofmembrane, filter, chip, or any other suitable solid support. Thepolynucleotides can be synthesized directly on the substrate, orsynthesized separate from the substrate and then affixed to thesubstrate. A microarray can be composed of a large number of unique,single-stranded polynucleotides, usually either synthetic antisensepolynucleotides or fragments of cDNAs, fixed to a solid support.

Hybridization assays based on polynucleotide arrays rely on thedifferences in hybridization stability of the probes to perfectlymatched and mismatched target sequence variants. For SNP genotyping, itis generally preferable that stringency conditions used in hybridizationassays are high enough such that nucleic acid molecules that differ fromone another at as little as a single SNP position can be differentiated(e.g., typical SNP hybridization assays are designed so thathybridization will occur only if one particular nucleotide is present ata SNP position, but will not occur if an alternative nucleotide ispresent at that SNP position). Such high stringency conditions may bepreferable when using, for example, nucleic acid arrays ofallele-specific probes for SNP detection.

In other embodiments, the arrays are used in conjunction withchemiluminescent detection technology.

A SNP detection kit/system of the present invention may includecomponents that are used to prepare nucleic acids from a test sample forthe subsequent amplification and/or detection of a SNP-containingnucleic acid molecule. Such sample preparation components can be used toproduce nucleic acid extracts (including DNA and/or RNA), proteins ormembrane extracts from any bodily fluids (such as blood, serum, plasma,urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin,hair, cells (especially nucleated cells), biopsies, buccal swabs ortissue specimens. The test samples used in the above-described methodswill vary based on such factors as the assay format, nature of thedetection method, and the specific tissues, cells or extracts used asthe test sample to be assayed. Methods of preparing nucleic acids,proteins, and cell extracts are well known in the art and can be readilyadapted to obtain a sample that is compatible with the system utilized.

Another form of kit contemplated by the present invention is acompartmentalized kit. A compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers include,for example, small glass containers, plastic containers, strips ofplastic, glass or paper, or arraying material such as silica. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the test samples andreagents are not cross-contaminated, or from one container to anothervessel not included in the kit, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother or to another vessel. Such containers may include, for example,one or more containers which will accept the test sample, one or morecontainers which contain at least one probe or other SNP detectionreagent for detecting one or more SNPs of the present invention, one ormore containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and one or more containers which containthe reagents used to reveal the presence of the bound probe or other SNPdetection reagents. The kit can optionally further comprise compartmentsand/or reagents for, for example, nucleic acid amplification or otherenzymatic reactions such as primer extension reactions, hybridization,ligation, electrophoresis (preferably capillary electrophoresis), massspectrometry, and/or laser-induced fluorescent detection.

Microfluidic devices, which may also be referred to as “lab-on-a-chip”systems, biomedical micro-electro-mechanical systems (bioMEMs), ormulticomponent integrated systems, are exemplary kits/systems of thepresent invention for analyzing SNPs. Such systems miniaturize andcompartmentalize processes such as probe/target hybridization, nucleicacid amplification, and capillary electrophoresis reactions in a singlefunctional device. Such microfluidic devices typically utilize detectionreagents in at least one aspect of the system, and such detectionreagents may be used to detect one or more SNPs of the presentinvention.

Further, the kits may also include one or more antigens, as describedabove. As such, the medical professional may first determine if a SNP ispresent or absent from the patient. Then, the medical professionalprepares a regiment, or uses instead a pre-determined regiment, to findwhich antigen, in what dose, by what method of administration, to givethe patient. The one or more antigens may be sold with, or sold separatefrom, the kit.

Given the disclosure in this application along with information wellknown to those of skill in the art, many SNPs that correlate with theability or difficultly of generating immune tolerance can be determined.PCR can be preformed on samples where the replicon contains these SNPsor where the replicon is closely linked to them on the genome. Suchtechnology can provide a rapid assay to determine the likelihood of theability to induce tolerance in a specific individual.

It should be apparent from the foregoing that an invention havingsignificant advantages has been provided. While the invention is shownin only a few of its forms, it is not just limited but is susceptible tovarious changes and modifications without departing from the spiritthereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Example 1 Rheumatoid Arthritis (RA) Patients

120 RA patients on maintenance conventional therapies for their RA weretested for the ability of their peripheral blood mononuclear cells(PBMC) to produce IFNγ when cultured for six days with the autoantigen,purified bovine α1 chain of Collagen II (CII) or α1(II) at 50 μg/u.Culture supernatants were harvested and IFNγ levels determined by ELISA.

${{IFN}\; \gamma \mspace{14mu} \alpha \; 1({II})\mspace{14mu} {S.I.}} = {\frac{{{IFN}\; \gamma \mspace{14mu} \alpha \; 1({II})} - {{IFN}\; \gamma \mspace{14mu} {PBS}}}{{IFN}\; \gamma \mspace{14mu} {PBS}} - 100.}$

As shown below (Table I), 76 patients had increased by two-fold overtheir unstimulated or (PBS) cultured PBMC, or a prevalence of 63% RApatients with an immune responses to CII (termed “Responders”). Althoughthere is a high prevalence of RA patients with CII autoimmunity, it isone of several possible antigens apparent during the disease.

TABLE I PERCENTAGE of RA PATIENTS WITH IMMUNE RESPONSE by CULTURED PBMCto CII* Patient N % Mean IFNγ α1(II) SI ± SEM Responders E 76 63 1494 ±313 Non-Responders 44 37 20.6 ± 6.5 Total 120 100 p = <0.001****Mann-Whitney Rank Sum Test

Example 2 Low Dose Collagen II Induces Tolerance in DBA/1 Lac Mice

Groups of 12 mice were fed oral CII at the doses indicated 8 times over2 weeks and immunized with 100 μg bovine CII in CFA. After 4 days rest,the mice were then immunized at the base of the tail with 100 μg CIIemulsified with complete Freund's adjuvant. The degree of arthritis wasassessed by a blinded observer for 8 weeks.

As can be seen in FIG. 6, 10 μg/day oral dose of CII was most effectivein reducing the incidence of arthritis with 500 μg/day being also, butless, effective. The percent of mice with grade 3 or 4 arthritis isindicated on the Y-axis. The biphasis response is due to induction ofdifferent tolerance mechanisms by low dose vs high dose CII. Low doseCII (10 μg) induces regulatory T cells while high dose (500 μg) inducesanergy or clonal deletion.

Example 3 NSAID Inhibition of Induction of Immune Tolerance to CollagenII in DBA/1 Mice

To determine whether tolerance induction to orally fed bovine CII inDBA/1 mice would be abrogated by orally fed NSAID, 3 groups of 20-22DBA/1 mice were fed (by gavage) eight doses (Monday, Tuesday, Thursday,and Friday for 2 weeks) of the following: Placebo (saline) in the a.m.and Placebo (0.1 M HAc) in the p.m.; Placebo (saline) in the a.m. and 10μg native bovine CII in the p.m.; or piroxicam (2.4 μg/gm) in the a.m.and native bovine CII (10 μg) in the p.m. After 1 week, all mice wereimmunized (intradermally at the base of tail) with 100 μg of bovine CIIemulsified in complete Freund's adjuvant. Animals were placed in codedcages and were scored by a blinded observer twice weekly for the numberof arthritic joints (joints swollen, red, and/or deformed).

As shown in FIG. 7, compared to Placebo+Placebo fed controls, thepercent of arthritic joints was less over the observation period in thegroup of mice fed Placebo+CII (p<0.03 by Cochran-Mantel-Haenszelanalysis). By contrast, there were significantly more arthritic jointsover the same period of observation in mice fed Piroxicam+CII. Insimilar studies, we found that nabumatone (Relafen) also abrogated OTinduction in DBA/1 mice (data not shown).

Example 4 IFNγProduction by Spleen Cells

To assess the effect of oral feeding of piroxicam to another group ofmice fed Placebo or CII on spleen cell IFNγ production in a mannersimilar to the experiment in FIG. 7, four groups of mice (4 mice pergroup) were gavaged 8 days over 2 weeks with the following: Piroxicama.m.-CII p.m.; Piroxicam a.m.-HAc p.m.; Saline a.m.-CII p.m.; or Salinea.m.-HAc p.m. After 1 week rest, all mice ere immunized at the base ofthe tail with a CII-complete Freund's adjuvant emulsion. After 14 days,mice ere sacrificed, and spleen cells were isolated and set up inculture with PBS or α1(II)CB peptide mixture. After 72 h culture,harvested supernatants ere analyzed for IFNγ levels by ELISA.

Mice fed CII+Placebo or piroxicam+Placebo had reduced production of IFNγwhen their spleen cells were cultured in vitro with α1(II) CB peptidemixture. See, FIG. 8. When piroxicam was orally fed to mice being fedoral CII, however, there was a dramatic increase in the level of IFNγproduction by spleen cells stimulated in vitro with α1(II)CB peptidemixture. In other experiments, it was found that the COX-2 inhibitorSC236 also blocked oral tolerance induction to CII in DBA/1 mice asassessed by IFNγ production by spleen cells (data not shown).

Example 5 COX-2 Inhibitor SC'236 Inhibits Oral Tolerance Induction

Groups of 8 mice each were gavaged on MTTHF×2 weeks in the a.m. with PBSor SC'236 (5 μg/gm in 100 μl PBS). SC'236 (Searle) is ˜2000× moreinhibitory for COX-2 than for COX-1.

Four of the mice given PBS in the a.m. and 4 mice given SC'236 in thea.m. were gavaged in the p.m. with 10 μg bovine type II collagen (CII).The other 4 mice in the a.m. PBS and a.m. SC'236 group were gavaged inthe p.m. with 0.1M acetic acid (HAc) the vehicle the CII was dissolvedin. After these 8 day feedings, all mice were rested for 1 week and thenimmunized with 100 μg of CII in complete Freund's adjuvant. After 10days, all mice were sacrificed, and spleen cells (2×10⁶/ml) wereisolated and set up in culture with PBS and bovine α1(II) (50 μg/ml) CBmixture. After 4 days culture, the supernatants were harvested and IFNγlevels determined by ELISA (Endogen). Since the PBS+spleen cell culturefrom all mice produced between 1-12 pg/ml IFNγ, only the α1(II) data areplotted. All groups were compared to Placebo control group by Student's2 sample t test.

As shown in FIG. 9, feeding CII to DBA/1 mice induced oral tolerancemanifested by a significant reduction in IFNγ production by spleen cellsstimulated by α1(II) CB digest (p<0.025). In contrast, feeding SC'236 tothe mice resulted in lower IFNγ production by α1(II) CB digest, but whenmice were fed SC'236+CII there was a significant increase (p<0.01) inIFNγ production by spleen cells cultured with α1(II) CB digest. Giventhe caveat that SC'236 may also inhibit COX-1, but to a degree ˜2000×less than it inhibits COX-2, these data suggest that COX-2 may beessential for optimal tolerance induction to low dose oral antigen.

Example 6 Persistent NSAID Effect on Oral Tolerance

To determine whether chronic feeding of piroxicam with CII would have apersistent effect on tolerance induction to CII in DBA/1 mice, threegroups of 10-11 DBA/1 mice were fed 8 doses of CII over two weeks (asabove) on two occasions separated by 6 months: piroxicam (2.4 μg/gm)a.m. and Placebo (0.1M HAc, 100 μl) p.m.; Placebo (saline) a.m. andnative bovine CII (10 μg) p.m; or piroxicam (2.4 μg/gm) a.m. and nativebovine CII (10 μg) p.m. Three months after the second 8 dose feeding,each mouse was immunized (intradermally at base of tail) with 100 μgnative bovine CII emulsified in complete Freund's adjuvant. Mice wereplaced in coded cages and scored twice weekly by a blinded observer fornumbers of arthritic joints. As shown in FIG. 10, at week 7 and 8, afterimmunization with CII, the group of mice that 9 and 3 months before werefed piroxicam plus CII had significantly more arthritic joints (p<0.04at 8 weeks by chi square analysis) compared to the group of mice thatwere fed Placebo plus CII.

Example 7 GALT of Mice Fed with Piroxicam Plus CII

To assess the status of the GALT in these three groups of mice, Peyer'spatch cells were isolated from each animal, and set up (2.5×10⁵/ml) inquadruplicate in co-culture with normal DBA/1 spleen cells (2×10⁶/ml)with addition of recombinant murine IL2 (10 U/ml). PBS (as a control) or50 μg/ml bovine α1(II) CB peptide mixture were added to quadruplicatewells in 96 round bottom well plates. After 3 days, cultures were pulsedwith ³H thymidine and harvested onto paper filters 24 h later. The“stimulation index” was calculated for each mouse by dividing the cpm ofα1(II) CB peptide mixture culture by the cpm of the PBS control culturefor each mouse. As shown in FIG. 11, compared to the co-culture ofPeyer's patch cells from mice fed CII alone or piroxicam alone, therewas marked stimulation by the α1(II) CB peptide mixture (p<0.03) ofcells from mice fed piroxicam plus CII. Moreover, we have consistentlyfound that Peyer's patch cells (2.5×10⁵/ml) from DBA/1 mice or Balb/cmice in co-culture with normal syngeneic spleen cells (2×10⁶/ml) whenstimulated with murine IL-2 results in no stimulation of the spleencells over background cpm. Thus, the marked increase in the stimulationindex of Peyer's patch cells with DBA/1 spleen cells in the presence ofα1(II) CB peptide mixture is quite exceptional and unexpected. Theculture of mesenteric lymph node cells (2×10⁶/ml) from each mouse withthe α1(II) CB peptide mixture revealed a similar pattern (FIG. 12). Themesenteric lymph node cells from mice fed either CII or piroxicam didnot proliferate in response to the α1(II) CB peptide mixture. Incontrast there was marked stimulation by α1(II) CB peptide mixture ofmesenteric lymph node cells from mice fed piroxican plus CII (FIG. 12).

Further, studies were conducted using oral OVA (1 mg/day×5 days) inBALB/c mice and oral bovine CII (10 μg×8 doses over 14 days) in DBA/1lac J mice to test the effects of commonly used immunomodulatory drugson immune induction (prednisone 7.5 mg/day, hydroxychloroquine 400mg/day, methotrexate 17.5 mg/week, leflunomide 20 mg/day after 100mg/day×3 loading doses, sulfasalazine 2.5 gm/day, D-penicillamine 750mg/day, IM gold, and etanercept 25 mg twice weekly). Theseimmunomodulatory drugs did not completely suppress tolerance, and thatit may be feasible to induce tolerance in RA patients to CII stilltaking these immunomodulatory drugs. Prednisone ≧10 mg/day equivalent inDBA/1 lac J mice and auranofin did block OT induction to CII.

Example 9 Administration of Oral Collagen II Reduces Autoimmunity inPatients with RA

Immunological tolerance, defined as a ≧30% reduction in IFN-γ productionby PBMC cultured with α1(II) of type II collagen, was examined inpatients. Patients that were taking disease-modifying antirheumaticdrugs, anti-TNF agents and/or non steroidal inflammatories wereadministered misoprostol 100 μg bid to reverse the non steroidalantiinflammatory inhibition of oral tolerance. Patients were randomizedto receive “low” or “high” doses of CII. The Low Dose group (n=38) tookdaily 30 μg/day bovine CII for 10 weeks, then 50 μg/day for 10 weeks andthen 70 μg/day for 10 weeks. The High Dose group (n=41) took 90 μg/dayfor 10 weeks, 110 μg/day for 10 weeks and then 130 μg/day for 10 weeks.

Heparinized blood was obtained at baseline and after each of the 10 weektreatment periods. The blood was diluted 1:3 with RPMI 1640 containingpenicillin (100 u/ml) and streptomycin (100 μg/ml) within 1-4 hoursafter collection, wrapped in paper, and placed in a styrofoam boxcontaining a “cold pack” and shipped overnight. The PBMC were isolatedfrom the blood samples and set up in culture with bovine α1(II) 25μg/ml, PHA 10 μg/ml or with 50 ul PBS. After 6 days in culture, cellfree supernatants were collected and stored at −70° for up to 7 monthsat which time all samples from a given patient were assayed for IFNγ bycommercial ELISA (R & D Systems). The results are shown as in FIG. 13.

The IFNγ stimulation index (SI) was calculated as

$\frac{{\alpha \; 1({II}){IFN}\; \gamma} - {{PBS}\mspace{14mu} {IFN}\; \gamma}}{{PBS}\mspace{14mu} {IFN}\; \gamma} \times 100.$

The SI for patients receiving each of the low doses (30 μg, 50 μg and 70μg/day) was compared with their SI at baseline before each of the 10week treatments and similarly with the high dose CII group (90 μg, 110μg and 130 μg/day) with their SI at baseline before each of the 10 weektreatments.

There was marked suppression of IFNγ SI. After ten weeks treatment with30 μg, 50 μg, 110 μg and 130 μg/day oral CII with ≧62 to 69% of patientsexhibiting ≧50% reduction in α1(II) IFNγ SI. 70 μg and 90 μg/day dosesdid not reduce the IFNγ SI, and the 70 μg/day dose significantlyincreased the IFNγ SI compared to baseline values.

Further, the data showed that oral tolerance to CII, defined as areduced immune response to fed antigen (CII), can be induced whilepatients are taking DMARDs, anti-TNF agents and NSAIDS if low dosemisoprostol is given. The 30 and 50 μg/day doses had greater reductionin more categories.

The dose response showed maximal suppression of IFNγ production at 30μg, 50 μg and 110 μg/day of oral CII. For the percent of patients thathad a 50% reduction in IFNγ by α1(II)-stimulated PBMC, most patientscould be tolerized (69% had a ≧50% reduction in α1(II) stimulated IFNγproduction to orally administered CII) at these doses.

Further analysis of patients revealed that there were 30% non-respondersto oral CII to the 30-50 μg/day dose of CII and 28% non-responders tothe 110-130 μg/day dose (FIG. 13).

Example 10 Oral CII Tolerance is Associated with Response and NoResponse

To investigate if any particular genotype was associated with responseor no response to oral CII in this cohort, 24 patients from Example 9were selected.

Blood was obtained from the patients before administering bovine CIIorally to RA patients who produced ≧2× increase in IFNγ by α1(II)stimulated compared to PBS control PBMC cultures. Isolated (10 μg/mL),bovine α1(II) (50 μg/mL), bovine α1(II) CB11 (50 μg/mL) or with 25 μlPBS added to culture at 2×10⁶ per 500 μl 48 well tissue culture platesof Dulbecos MEM supplemented with penicillin (100 μg/mL), streptomycin(100 μg/mL) and 9% fetal calf serum. After 6 days, supernatants wereharvested, centrifuged at 2000×G for 5 minutes and levels of IFNγ werequantitiated by commercial ELISA (R & D Systems). Differences in IFNγlevels and in SI for PHA, α1(II) and α1(II) CB11 between responders andnon-responders were analyzed for significance using Mann-Whitney ranksum test.

Of the 24 patients, 16 patients responded to oral CII with reduction ofα1(II) and α1(II) CB11 stimulated IFNγ production by PBMC cultures and 8patients did not reduce IFNγ by PBMC culture with these antigens. Thebaseline IFNγ levels of the 16 responders and 8 non-responders to PBS,α1(II) 50 μg/ml α1(II) CB11 50 μg/ml and PHA 10 μg/ml in six day PBMCcultures are given in Table I.

The IFNγ stimulation index (S.I.) calculated for PHA as follows:

${\frac{{{PHA}\mspace{14mu} {IFN}\; \gamma} - {{PBS}\mspace{14mu} {IFN}\; \gamma}}{{PBS}\mspace{14mu} {IFN}\; \gamma} \times 100};$

for α1(II) as follows:

${\frac{{\alpha \; 1({II}){IFN}\; \gamma} - {{PBS}\mspace{14mu} {IFN}\; \gamma}}{{PBS}\mspace{14mu} {IFN}\; \gamma} \times 100};$

and for α1(II) CB11 as follows:

$\frac{{\alpha \; 1({II}){CB}\; 11\mspace{14mu} {IFN}\; \gamma} - {{PBS}\mspace{14mu} {IFN}\; \gamma}}{{PBS}\mspace{14mu} {IFN}\; \gamma} \times 100.$

As shown in Table II, the CII oral tolerance non-responders had lowermean baseline IFNγ α1(II) S.I.s than the CII oral tolerance responders(190±40 vs 1800±520, p=0.002). CII oral tolerance non-responders hadlower mean baseline IFNγ α1(II) CB 11 S.I.s than the CII oral toleranceresponders (1060±197 vs 210±90, p=0.003). The IFNγ PHA S.I.s at baselinewere not different between the CII oral tolerance responders andnon-responders.

TABLE II Comparison of IFNγ Production at Baseline Between CII OTResponders and Non-Responders CII OT Responders CII OT Non-Responders (N= 16) (N = 8) Culture IFNγ IFNγ Adds PG/mL IFNγ S.I. PG/mL IFNγ S.I. PBS142 ± 41 — 203 ± 159 — (p = 0.395) α1(II) 1774 ± 501 1800 ± 520 581 ±183 190 ± 40 (p = 0.150) (p = 0.002) α1(II) 1391 ± 295 1060 ± 197 523 ±167 210 ± 90 CB11 (p = 0.06) (p = 0.003) PHA  6979 ± 2291  6400 ± 31003150 ± 1278  4200 ± 3200 (p = 0.520) (p = 0.349)

Example 11 The Microarray Assessment Produced Accurate Data for Analysisof Genotypes of RA Patients

SNP analysis was performed of 16 responders and 8 non-responders to oralCII to find the frequency of SNPs closely associated on chromosomes nextto several cytokines and chemokines known to be important for oraltolerance induction. The 16 patients were those with had ≧50% reductionin IFNγ al stimulation index from baseline to either the 30 μg/day, 50μg/day, 110 μg/day or 130 μg/day dose of CII (“OT Responders”). The 8patients with increases in IFNγ α1S.I. at the 30 μg/day, 50 μg/day, 110μg/day or 130 μg/day doses of oral CII were selected (“OTNon-Responders”).

Commercial whole genome mapping chips were used to map the potentialgenetic loci in a timely and economic manner. The genome of the twogroups of Example 2 were analyzed by DNA extraction using a commercialDNA extraction kit, the Qiagen kit (Qiagen Inc., Alameda, Calif.)following the manufacturer's instructions. After determining the qualityand quantity of the DNA in an Eppendorf photometer (Eppendorf ScientificInc., Westbury, N.Y.), and by electrophoresis, DNA with OD260/280 ratiosratio>1 and high integrity was used for genotyping. For each sample, 250ng of DNA was used for genotyping using Affy GeneChip® Mapping 10K 2.0Array, a SNP-based genetic mapping tool. The 10K 2.0 Array containsgenotypes greater than 10,000 human single nucleotide polymorphisms(SNPs) on a single array. The tool may be used to identify regions ofthe genome that are linked to or associated with, a particular trait orphenotype, in our case, the CII oral tolerance resistance.

The protocol included four major procedures: in silico fractionation,synthesis of predicted fragments on microarrays, biochemicalfractionation, and Allele specific hybridization and Genotype Calling.Two different signals that represent each of two polymorphisms of 10,000single nucleotides were produced. The software creates the polymorphismor genotype of every of 10,000 single nucleotides. The genotype ofpolymorphism of each of 10,000 SNPs of every sample was called intothree types, homozygous type I, AA; homozygous type II, BB; andheterozygous, AB, as indicated in table III.

TABLE III Genotyping of 15 Patients Called Signal Patient # Gender SNPCall Detection AA Call AB Call BB Call 001-101 M 94.41% 99.67% 33.27%32.90% 33.83% 023-117re M 93.70% 99.59% 33.54% 32.89% 33.56% 046-123 F78.89% 99.25% 32.53% 35.37% 32.10% 073-127 M 93.57% 99.40% 34.21% 31.30%34.49% 090-323 F 95.87% 99.89% 32.21% 33.99% 33.80% 095-325 F 94.57%99.05% 32.66% 33.82% 33.51% 109-319 F 85.35% 97.53% 34.89% 30.66% 34.45%121-329 M 93.44% 99.55% 33.70% 32.28% 34.02% 127-137 M 93.82% 99.57%33.35% 33.30% 33.34% 142-343 M 94.93% 99.80% 34.12% 31.86% 34.02%143-339 M 96.28% 99.92% 32.76% 34.00% 33.25% 146-139 M 92.07% 99.05%33.45% 32.09% 34.45% 148-341 F 91.31% 99.18% 33.38% 33.21% 33.41%154-347 F 96.89% 99.95% 33.06% 34.18% 32.76%

Table III is the genotyping of 15 patients (9 males, 6 females). Thedetectable single of SNPs in most samples was over than 99%, indicatinga high quality of detection. The SNP call indicates that the percentageamong 10,000 of SNPs that could be recognized and for which data weregiven. More than 90% of SNPs in those samples was detected by theexperiment. The distribution of three genotypes, the AA, AB, and BB wasabout one third (33%) for each of them.

Example 12 Association Between SNP_A-1515737 and Oral CII Responders andNon-Responders

To find the possible polymorphism that linked non-tolerogenic responseto CII treatment, the genotype of the patients of Example 3 was sortedinto oral CII responders and non-responder groups. For each group, thetotal number of three genotypes, AA, AB, and BB was calculated for eachSNP on the chip. Table IV shows the genotype ratio of SNP in each ofthose candidate genes.

TABLE IV Polymorphism of Candidate Genes. AA-AB-BB Candidate Gene nameSNP IDs (responder/non-responders) TGF beta 1, 2 3 SNP_A-15111173--4-6/3--2--4 SNP_A-1515879 8--6-0/9--1--0 ICOS SNP_A-15091143--4-5/0--8--2 SNP_A-1509255 6--7-1/2--6--2 SNP_A-1513931 13-1-0/9--1--0SNP_A-1515899 0--0-14/0--0--10 SNP_A-1519289 2--1-7/2--4--4 IL-4RSNP_A-1509275 3--9-1/7--3--0 IL-10R SNP_A-1518241 2--3-7/4--3--2 CCL2SNP_A-1514598 1--5-8/3--4--3 IFN gamma SNP_A-1508498 8--5-1/8--2--0SNP_A-1512645 3-11-2/0--4--4 SNP_A-1512719 2-10-2/5--5--0 SNP_A-15153303--9-2/5--4--0 SNP_A-1515737 1--9-6/6--1--1 SNP_A-1518829 2-10-4/0--2--6SNP_A-1518878 3-10-1/7--2--1 Glutamic Acid SNP_A-1513856 14-0-0/10--0-0decarboxylse SNP_A-1509772 2--8-4/1--6-3 IL-1ra SNP_A-15112802--5-7/0--3--7

As shown in Table IV, in 20 SNPs of 8 candidate genes, the mostgenotypic patterns between responder and non-responders are the same orsimilar. The patterns of the first SNP of Glutamic Acid decarboxylse is14-0-0 (for AA-AB-BB) and 10-0-0; the second SNP is 2-8-4 and 1-6-3.However, there is a large difference of genotype distribution of theSNP_A-1515737 between response and non-response groups, being 1-9-6 and6-1-1 respectively for responders and non-responders. While most ofresponse patients have genotype either heterozygous (thus, the AG) orhomozygous of BB (Thus the GG genotype), the majority of patients innon-response group had a AA genotype. Further analysis focused on theSNP_A-1515737.

The sequences of SNP_A-1515737 is as the following:TTTTTTTTTTGTACCT[A/G]GTTCTATGGTTACCTT (SEQ ID NO. 1 and 2). The A/G isthe polymorphic site. Thus, AA represents AA homozygous, while ABrepresents A/G heterozygous and BB represents GG genotype, A→Grepresents the polymorphism site.

Example 13 Distribution of SNP_A-1515737 Among CII Oral ToleranceResponder and Non-Responder Patient

Genotype patterns of responders and non-responders were compared and asignificant difference in the genotype distribution found. See, FIG. 14.To calculate the values, a number was assigned to the three genotypes.The AA genotype was assigned 1, AB (thus, AG) was assigned 2, and the BB(Thus GG genotype) was assigned 3. According to those assumptions, the Pvalues of SNP_A-1515737 reached 0.052.

The 24 patients were grouped into those had the AA genotype (or A→G) ofSNP_A-1515737 and those that did not have the AA genotype (i.e. were ABor BB). Table V and VI summarize the data based on the two groups. TableV lists the baseline IFNγ levels in six day supernatants PBMC stimulatedfor six days with PHA, α1(II), α1(II) CB11 or no additions (PBS).Patients with SNP_A-1515737 AA had significantly lower IFNγ α1(II) S.I.values (mean 270±90), compared to those patients not having threeSNP_A-1515737 AA (mean 1674±505, p=0.028 by Mann-Whitney Rank Sum Test).Of the 7 patients with SNP_A-1515737 AA only one had CII oral toleranceresponse and of the 17 patients who have SNP_A-1515737 GG only 2 morenon CII oral tolerance responders.

TABLE V Baseline IFNγ (PG/ML) in 6 Day PBMC Culture Supernatants No ofSNP A-1515737 AA Genotype α1(II) Patient Geno- PHA α1(II) α1(II) CB11 OT# type PHA SI A1(II) SI CB11 SI PBS Responder 073-127 BB 16278 2533 39376052 4574 7047 234 Yes 001-101 AB 486 817 1348 2443 292 451 53 Yes023-117 AB 14638 47119 256 726 389 1155 31 Yes 039-303 AB 446 1012 98.5146 51 27 40.1 No 046-123 BB 7679 1474 1474 203 486 -0.4 488 No 090-323BB 1163 2054 1483 2646 1451 2587 54 Yes 109-319 BB 4444 11443 61 58 123219 38.5 Yes 121-329 AB 6983 3017 1728 671 2243 901 224 Yes 127-137 BB618 301 505 228 500 225 154 Yes 143-339 AB 1056 1752 4188 7247 1273 213357 Yes 142-343 AB 189 133 325 301 383 373 81 Yes 148-341 BB 23427 88761019 290 1523 484 261 Yes 146-139 AB 1431 3821 630 1626 1271 3382 36.5Yes 154-347 AB 3774 6521 284 398 843 1379 57 Yes 072-128 BB 2755 3007688 1017 2205 220 688 Yes 113-332 AB 245 113 2641 2197 1865 1522 115Yes 040-302 AB 29527 64089 1058 2200 433 841 46 Yes 6723 ± 9140 ± 1690 ±1674 ± 1171 ± 1350 ± 146 ± 2165 4373 481 505 274 425 94 SNP A-1515737 AAGenotype α1(II) Patient Geno- PHA α1(II) α1(II) CB11 OT # type PHA SIα1(II) SI CB11 SI PBS Responder 097-320 AA 273 82 1208 265 1268 280 331No 050-308 AA 707 1314 90 80 120 140 50 No 058-309 AA 2093 776 455 90294 23 239 No 061-311 AA 3173 1256 671 187 1240 430 234 No 095-325 AA1744 1103 1228 747 2894 1896 145 Yes 155-348 AA 1050 405 461 122 428 106208 No 014-109 AA 9777 26686 182 399 293 703 36.5 No 2688 ± 4517 ± 614 ±270 ± 934 ± 511 ± 245 ± 1236 3699 172 90 371 247 40 p = p = p = p = p =p = p = 0.546 0.240 0.172 0.028 0.391 0.153 0.070

In Table VI, the same patients were arranged and their IFNγ α1(II) S.I.at baseline and after oral CII administration (30 μg, 50 μg or 110μg/day for 10 weeks) was compared. As shown in Table VI, patients whodid not have SNP_A-1515737 AA (ROT1 AA) genotype had a significantreduction in the IFNγ α1(II) S.I. after oral CII compared to baselinevalues (p<0.001 by Wilxocon Rank Sum Test). In contrast, patients whocarried the SNP_A-1515737 AA had no significant change in IFNγ α1(II)S.I. after oral CII treatment (p=0.230 by Wilcoxon Rank Sum Test). Thiswas also reflected when data are represented as a ratio of IFNγ α1(II)S.I. after oral CII/baseline (p=0.011).

TABLE VI REDUCTION IN α1(II) SI AFTER ORAL CII IN PATIENTS WITH RAPatient α1(II) SI After Oral OT Ratio: # Genotype Baseline CII ResponderAfter/baseline No SNP A-1515737 AA Genotype 073-127 BB 6065 18 Yes0.0030 001-101 AB 2443 246 Yes 0.1008 023-117 AB 726 360 Yes 0.4932039-303 AB 146 280 No 1.9178 046-123 BB 203 590 No 2.9064 090-323 BB2646 152 Yes 0.0573 109-319 BB 58 6 Yes 0.1034 121-329 AB 671 276 Yes0.4119 127-137 BB 228 18 Yes 0.0789 143-339 AB 7247 503 Yes 0.0694142-343 AB 301 19 Yes 0.0631 148-341 BB 290 0 Yes 0 146-139 AB 1626 3Yes 0.0018 154-347 AB 398 7 Yes 0.0176 072-128 BB 1017 358 Yes 0.3520113-332 AB 2197 93 Yes 0.0423 040-302 AB 2200 540 Yes 0.2454 2081 ± 505 175 ± 46  0.427 ± 0.217 p = 0.011 p ≦ 0.001 SNP A-1515737 AA Genotype097-320 AA 265 630 No 2.3774 050-308 AA 80 404 No 5.0500 058-309 AA 90268 No 2.9778 061-311 AA 187 2020 No 10.8021 095-325 AA 747 3 Yes 0.0040155-348 AA 122 167 No 1.3688 014-109 AA 399 800 No 2.0050 270 ± 90  613± 256 3.512 ± 1.347 p = 0.230

The data was analyzed by use of Chi Square with Fisher's exact test,there is a highly significant difference (p=0.0017) between oral CIIresponders and non-responders in patients with SNP_A-1515737 AA genotypeand those not having this genotype (Table VII).

TABLE VII Chi Square of SNP A-1515737 AA Genotype vs Non SNP A-1515737AA Genotype AA Non-AA CII OT Non-Responder 7 2 CII OT Responder 1 15

Example 14 Differences Between Responders and Non-Responders SNPs

The genotype patterns of other SNPs within close distance to A-1515737was also examined. In addition to A-1515737, there are six other SNPwithin the same genome region. None of them has a significantassociation with the response or non-response to CII treatment. Forexample, SNP_A-1508498, which is 146068 by at 5′ side of DYRK2 and350588 by at 3′ side: IFNγ, is located very close to A-1515737, which is265143 by at 5′ side of DYRK2 and 231513 by at 3′ side of IFNγ. Thegenotype patterns of responders and non-responders are similar, with AA,AB, and BB of 8-5-1 and 8-2-0, respectively.

The segregating patterns of an average of 10 SNP along each chromosomewas examined, but there was no evidence of an association of anysegregating pattern with the CII response.

Eight SNPs relevant to the non-response histocompatibility complex (HLAclass II histocompatibility) was examined but there was no evidence ofan association of the segregating bands with the CII response.

Example 15 SNP_A-1515737 and Linkage to Oral Tolerance Resistance inOther Autoimmune Diseases

Samples from 26 patients from a trial of oral type I collagen (CI) inpatients with diffuse systemic sclerosis (SSc) who took 500 μg/day CIfor 12 months were collected. Six of the 26 had A-1515737 AA genotype(Table VII). No patients were on DMARDs, biologies, NSAIDS orprednisone.

PBMCs were collected from the patients and cultured with native bovineCI and α2(I) CB mixture and a trend toward defective production IL-10PBMC cultured with α1(I) CB mixture.

Patients who were tolerized by oral CI had upregulation of the Th2cytokine, IL-10 by PBMC stimulated with CI, or α1(I) or α2(I). As shownin table VII, SSc patients with ROT1AA genotype and did not upregulateIL-10 production by α2(I) or CI stimulated PBMC in SSc patients after 12months of treatment with oral CI. THE SNP A-1515737 was also examined in53 additional SSc patients and found the overall prevalence of AA was32%, 35%

TABLE VII IL-10 PRODUCTION CHANGE AFTER 12 MONTHS OF ORAL CI and 12MONTHS FOR SSc PATIENTS WITH and WITHOUT ROT1 AA* Bovine Native CI IL-10pg/ml Genotype AA Patient # N = 6 −825 ± 338 Genotype BB or BA Patient #Mann Whitney   27 ± 97 Rank Sum Test p = 0.008 Fisher's Exact Test p =0.014 were GG and 30% were GA. Like in RA patients, SSc patients with AAexhibited less upregulation of IFNγ production by α2(I)-stimulated PBMC(Tables VII and VIII).

TABLE VIII IFNγ Production by Hα1(I)-Stimulated PBMC from SSc Patientsat Baseline # of Patients IFNγ pg/ml ROT1AA+ 25  16 ± 134 ROT1GG or GA54 3146 ± 2574 Mann Whitney Rank Sum Test p = 0.036 * PBMC of patientsenrolled in Phase II CI/SSc Study were cultured with 25 μg/ml humanα1(I) for 6 days after which culture supernatants were harvested andIFNγ levels determined by ELISA.

Example 16 IL-10 Production After 12 Months of Oral CI and 12 Months forSScPatients with and without ROT1 AA

Patients with ROT1 AA, GG or GA genotype had IL-10 measured of IL-PBMCcultures stimulated by α1(I) CB mixture, α2(I) CB mixture, or native CIafter receiving oral CI for 12 months minus the values of IL-10 in thesupernatants of PBMC cultures stimulated with the same antigens atbaseline before oral CI was administered to the SSc patients.

Patients with ROT1 AA genotype received oral bovine CI 500 ug/day for 12months had deficient upregulation of IL-10 production by their PBMC whencultured with native bovine CI and α2(I) CB mixture and a trend towarddefective production IL-10 PBMC cultured with α1(I) CB mixture,demonstrating that ROT1 AA is associated with impaired oral tolerance toprotein antigen. See, Table IX.

TABLE IX IL-10 PRODUCTION CHANGE AFTER 12 MONTHS OF ORAL CI and 12MONTHS FOR SCLEROSIS PATIENTS WITH and WITHOUT ROT1AA* Bovine α1(I) CBBovine α2(I) CB Mix Mix Bovine Native CI IL-10 pg/ml IL-10 pg/ml IL-10pg/ml Genotype AA Patient # 071109 −342 −1692 −1726 130403 −19 +28 +70061009 −611 −494 −920 060507 −112 +490 −233 060403 −2942 −3736 −1886040104 +295 −448 −253 −622 ± 480 −975 ± 627 −825 ± 338 Genotype GG or GAPatient # 020705 −314 −388 −135 030504 +410 +203 +566 070101 −104 −86−334 080101 −391 −180 +192 011008 +529 +859 +103 021308 +216 +546 +386040806 −968 −1000 −1564 020906 +347 +261 +233 021411 −397 −2021 +21041308 +1364 +1460 +237 072317 +108 +145 +150 061211 −205 −113 −94072014 −81 +141 +32 090605 −561 −509 −282 091310 +582 +5 +232 030103 −46+381 +192 030201 −538 +252 −52 050303 −227 −115 +43 072418 +663 −415+269 080403 +221 +141 +341 Mann Whitney    30 ± 119    22 ± 156   27 ±97 Rank Sum Test p = 0.062 p = 0.039 p = 0.008 Fisher's NS NS p = 0.014Exact Test

Example 17 ROT1 Genotype in Patients and Family Members with Chron'sDisease

We assessed SNP_A-1515737 (ROT1) genotype in patients and/or familymembers with Crohn's and/or ulcerative colitis and healthy controls withno IBD or other known autoimmune disease. Table X summarizes thegenotype results in Crohn's diseases using DNA from buccal swabs,showing the ROT1 AA distribution was 91.67% for patients with Chron'sDisease.

TABLE X ROT1 Genotype Total (%) Distribution of ROT1 Genotypes inPatients with Definite Crohn's Disease AA 8 AG 0 GG 0 Distribution ofROT1 Genotypes in First Degree Relatives of Patients with Crohn'sDisease or Crohn's plus Ulcerative Colitis AA* 9 AG 0 GG 0 *Relativeshave CD or CD and UC

Of all the Crohn's disease patients and their first degree relativesgenotyped, 91.67% have ROT1AA. This evinces that ROT1AA is associatedwith oral tolerance resistance in Crohn's disease.

The prevalence of ROT1AA in 12 Crohn's disease and first degreerelatives (91.6%) was greater than 79 patients with SSc (31.6%), 54patients with RA (38.9%) and in healthy controls (35.7%) (See Table XI).

TABLE XI Distribution of ROT1 Genotype in Patients with SystemicSclerosis, Rheumatoid Arthritis and Healthy Controls Systemic RheumatoidNormal ROT1 Sclerosis Arthritis Controls Genotype Total Percent TotalPercent Total Percent AA 25 31.6% 21 38.9% 5 35.7%  AG 24 30.3% 17 31.5%6 42.86% GG 30 38% 16 29.6% 3 21.43%

Example 18 ROT1 Genotype is Present in 31% of Patients with Diffuse SSc

Banked PBMC cell pellets were surveyed for all the patients with diffuseSSc, and 32% were found to be homozygous for ROT1 AA, 35% werehomozygous for ROT1 GG, and 30% were heterozygous ROT1 GA.

The SSc patients with ROT1 AA exhibited less upregulation of IFNγproduction by α1(I)-stimulated PBMC. In the Phase II oral CI toleranceclinical trial at 12 months, 79 SSc patients were genotyped of the 168enrolled in the clinical trial. Seven of the 23 ROT1AA genotype were inthe Late Phase category, and deleted from the results. Reanalysis of thecompleters shows a significant difference in the change in MRSS at 12months from baseline values in the CI treated patients when compared tothe placebo treated patients using the Wilcoxon Rank Sum test (see FIG.16).

TABLE 1 Antigen Disease 1D protein Endocrine orbitopathy 59-kD renalantigen Progressive glomerulonephritis Acetylcholine receptor subunitMyastenia gravis Aggrecan, fibrillin and Juvenile idiopathic arthritismetalloproteinase-3 Alpha3 (IV) NI1 Anti-GBM disease Alpha-enolaseAsthma Alpha-fetoprotein Juvenile Batten disease Annexin A6 Neonatallupus erythematosus Apoptotic cell-binding protein Systemic lupuserythematosus AUF1 Systemic rheumatic diseases Autologous colonextracted Crohn's disease proteins Beta2-glycoprotein-I Antiphospholipidsyndrome Blood cell autoantigen Autoimmune hemolytic anemia Borreliaburgdorferi lysine- Lyme encephalitis enriched protein Borrelia T cellepitope Lyme arthritis BP180 Bullous pemphigoid BPAg2 IgA disease C1DPolymyositis-scleroderma overlap syndrome Collagen, preferably Type VIdiopathic pulmonary fibrosis collagen Cytochrome P450 1A2 Hepatitisgraft-versus-host disease Cytokeratin-10 Lyme arthritis Deamidatedgliadin peptide Celiac disease Desmocollin 1 Pemphigus Desmoglein 1Pemphigus foliaceus Desmoglein 1 and 3 Pemphigus Desmoglein 1 and 3Pemphigus vulgaris Desmoglein 1 and 3 Pustular dermatosis(Sneddon-Wilkinson disease) Desmoglein 3 ectodomain Pemphigus vulvarisDesmoglein-3 peptides Pemphigus vulgaris Enolase and arrestin Multiplesclerosis GAD65 Type 1 diabetes Glatiramer acetate Multiple sclerosisGlobular domain of human C1q Systemic lupus erythematosus GlutathioneS-transferase theta 1 Primary sclerosing cholangitisGPI-anti-oxLDL/beta2GPI SLE Heat shock proteins Carotid atherosclerosisHeparin Thrombocytopenia Histidyl-transfer RNA synthetase Jo-1autoantibody-associated myositis hnRNP A/B proteins Systemic rheumaticdiseases and hnRNP L hnRNP-A2 (RA33) Pristane-induced arthritis HRES-1endogenous retrovirus Systemic lupus erythematosus Hsp60 Juveniledermatomyositis hsp60, -65 and -70 Juvenile idiopathic arthritis Humaninsulin Diabetes mellitus type I Human intestinal antigens Crohn'sdisease Intrinsic factor Autoimmune gastritis Jo-1 or Ro-52/Ro 60Myositis patients Ku Connective Tissue Lens proteins Uveitis MBL RAMegalin Donnai-Barrow and faciooculo- acoustico-renal syndromes Myelinbasis protein peptides Multiple Sclerosis NeurofascinAutoantibody-mediated axonal injury Neuron-specific enolase Suddenacquired retinal degeneration syndrome Noncollagenous 1 and 2 domainsChildhood epidermolysis bullosa of type VII collagen acquisitaNoncollagenous domain of type Herpes gestationis VII collagen Nuclearribonucleoprotein A2 Systemic lupus erythematosus (hnRNP-A2) Nucleosomeantigen Lupus erythematosus Nucleosomes Lupus nephritis p200 Pemphigoidp200 pemphigoid antigen Epidermolysis bullosa acquisita Parotid antigensSjögren's syndrome Pemphigus vulgaris IgG Acantholysis Phenylalanyltransfer RNA Polymyositis synthetase Plasma membrane autoantigensAutoimmune hepatitis Poly (ADP-ribose) polymerase 1 Systemic lupuserythematosus Rabaptin 5 as a novel autoantigen Alzheimer's disease RAPand megalin Heymann nephritis Recombinant 70 kDa Mixed connective tissuediseases ribonuceloprotein Red blood cells as model antigensAutoimmunity Retinal antigen Macular degeneration Retinal SolubleAntigen Uveitis Ribosomal P protein Systemic lupus erythematosusRibosomal P Protein PO Mixed connective tissues RNA helicase A Systemiclupus erythematosus Selenium binding proteins Behcet's disease SmSystemic lupus erythematosus Smd 183-119-autoantigen SLE Spinal cordcells Amyotrophic lateral sclerosis (ALS) Squamous Cell Carinoma AntigenPsoriasis Protein Family SSA/SSB antibodies Systemic lupus erythematosusSubunit of RLIP76 Immune-mediated vascular diseases and atherosclerosisTestis-expressed protein TSGA10 Autoimmune polyendocrine syndrome type ITG3 Celiac disease Thyroid hormone Autoimmune encephalopathy Thyroidperoxidase Type 1 diabetes Transglutaminase Dermatitis herpetiformis andceliac sprue TRIM proteins Sjögren syndrome Type I collagen Systemicsclerosis (Scleroderma) Type I collagen Idiopathic Pulmonary FibrosisType II collagen RA Type III collagen Systemic sclerosis (Scleroderma)Type III collagen Idiopathic Pulmonary Fibrosis Type V collagenIdiopathic Pulmonary Fibrosis Vimentin RA ZnT8 (Slc30A8) Human type 1diabetes

1. A method for screening for susceptibility to immune tolerancedevelopment, comprising screening for at least one SNP.
 2. A method ofclaim 1, wherein said method of screening comprises FISH.
 3. The methodof claim 1, wherein said method of screening comprises use of a DNAarray.
 4. The method of claim 1, wherein said method of screeningcomprises hybridizing a polynucleotide probe.
 5. The method of claim 4,wherein said method is selected from the group consisting of:allele-specific probe hybridization, allele-specific primer extension,allele-specific amplification, sequencing, 5′ nuclease digestion,molecular beacon assay, oligonucleotide ligation assay, size analysis,and single-stranded conformation polymorphism.
 6. The method of claim 1,wherein said method comprises correlating the presence or absence of theat least one SNP with ability of a host to develop immune tolerance as aresult of administration of one or more antigens to the host.
 7. Themethod of claim 6, wherein said host is a human.
 8. The method of claim6, wherein said at least one or more therapeutic agents comprises atleast one antigen.
 9. The method of claim 8, wherein said at least oneantigen is a collagen.
 10. The method of claim 8, wherein said collagenis selected from the group types consisting of: I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX,XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII and XXVIII.
 11. A method ofscreening for susceptibility of a host to immune tolerance development,said method comprising: a. obtaining a sample from said host comprisingnucleic acid; b. isolating nucleic acid from said sample; c. assayingsaid sample for the presence or absence of at least one SNP, wherein thepresence or absence of the at least one SNP is indicative of anincreased susceptibility to develop immune tolerance.
 12. The method ofclaim 11, wherein said host is human.
 13. The method of claim 11,wherein said sample is selected from the group comprising: whole blood,blood plasma, urine, tears, semen, saliva, buccal mucosa, interstitialfluid, lymph fluid, meningeal fluid, amniotic fluid, glandular fluid,sputum, feces, perspiration, mucous, vaginal secretion, cerebrospinalfluid, hair, skin, fecal material, wound exudate, wound homogenate, andwound fluid.
 14. The method of claim 11, wherein said immune toleranceis induced by administration of at least one antigen to the host. 15.The method of claim 14, wherein said at least one or more antigens is acollagen.
 16. The method of claim 15, wherein said collagen is selectedfrom the group types consisting of: I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII,XXIII, XXIV, XXV, XXVI, XXVII and XXVIII.
 17. The method of claim 11,wherein said immune tolerance developed is to a sclerotic disease. 18.The method of claim 17, wherein said sclerotic disease is systemicsclerosis.
 19. The method of claim 11, wherein said method furthercomprises at least one computer program for use with at least onecomputer system, wherein said computer program includes a plurality ofinstructions comprising: a. at least one instruction for aiding inidentification of the presence or absence of said at least one SNP; b.at least one instruction for associating the presence or absence of saidat least one SNP with at least one disease state; and c. at least oneinstruction for correlating the presence or absence of said at least oneSNP with a score indicating susceptibility of a host to develop immunetolerance.
 20. The method of claim 19, wherein said computer programfurther generates a report comprising the results of the plurality ofinstructions.
 21. The method of claim 20, wherein said report istransmitted over a network.
 22. The method of claim 20, wherein saidreport is transmitted through an on-line portal.
 23. The method of claim20, wherein said report is transmitted by paper or e-mail.
 24. Themethod of claim 20, wherein said report is transmitted in a securemanner.
 25. The method of claim 20, wherein said report is stored in adatabase.
 26. A method of administering at least one therapeutic agent,the method comprising a. genotyping one or more SNP(s) in the nucleicacid of a host, b. correlating the one or more SNP(s) with one or morediseases or disorders, c. using a mathematical algorithm to determineprobability that said host will respond positively or negatively toadministration of at least one therapeutic agent, and d. administratingor not administrating a therapeutic agent to the host based on theresults of said mathematical algorithm.
 27. A method for conducting aclinical trial in which one or more antigen(s) are evaluated, saidmethod comprising: a. genotyping one or more SNP(s) relating to one ormore diseases or disorders; b. analyzing the genotyping results; c.determining a course of action based on the results of said genotyping,wherein said course of action comprises i. including individual in theclinical trial based on the results of said genotyping having indicatedthat said individuals are likely to respond to said one or moreantigen(s), and/or ii. excluding individuals from participating in theclinical trial based on the results of said genotyping having indicatedthat said individuals are not likely to respond to said one or moreantigen(s).
 28. A method for identifying an individual who has analtered risk for developing an autoimmune disease, comprising detectinga single nucleotide polymorphism (SNP) in SEQ ID NO: 1 in saidindividual's nucleic acids, wherein the presence of the SNP iscorrelated with an altered risk for autoimmune disease.